Neurogenesis inducing genes

ABSTRACT

The present invention relates to neurogenesis inducing genes. In particular, the present invention provides neurogenesis inducing genes coding for Zic proteins, vectors containing such genes, host cells containing such vectors, proteins produced by such host cells, antibodies raised to such proteins, and therapeutic agents or agents for gene therapy for nervous diseases.

This application is a Divisional of U.S. patent application Ser. No.09/342,325, filed Jun. 29, 1999, now U.S. Pat. No. 6,500,637, which is aContinuation-In-Part of U.S. patent application Ser. No. 09/172,045,filed Sep. 28, 1998, now U.S. Pat. No. 6,277,594, which claims prioritybenefit to Japanese Patent Appln. Nos. 86979/1998, filed Mar. 31, 1998,and 121456/1998, filed Apr. 30, 1998.

FIELD OF THE INVENTION

The present invention relates to neurogenesis inducing genes.

BACKGROUND OF THE INVENTION

The early process of vertebrate neurogenesis is divided into severalbasic processes, such as differentiation into the neural plate (neuralinduction) and formation and maturation of the neural network from theectoderm. This early process includes occurrence of neural precursorcells, pattern formation of the nervous system, and proliferation anddifferentiation of neural precursor cells.

It is known that the early neurogenesis of Xenopus laevis is induced byblockade of BMP4 (i.e., Bone Morphogenetic Protein 4) signals by noggin,chordin, etc. (Sasai et al., Nature, 376:333 [1995]; and Mizuseki etal., Development 125:579-587 [1998]). BMP4 is a factor which induces theectoderm into epidermal cells. When BMP4 is activated, cellsdifferentiate into the epidermis. Proneural genes (e.g., Neurogenin,NeuroD, XASH-3, XATH-3) which are involved in the control of neuralinduction (i.e., neurogenesis, neural differentiation) and in the codingfor basic helix-loop-helix (bHLH) transcription factors are also known.However, the factors involved in the blockade of BMP4 signals toproneural genes are still unknown.

Understanding the molecular basis of higher brain functions isimportant, not only to elucidate the universal principle of theseprocesses, but also in the development of new therapeutic methods fortreatment of diseases involving brain functions (i.e., neurogenesis).

SUMMARY OF THE INVENTION

The present invention provides neurogenesis inducing proteins, genescoding for the proteins; recombinant vectors comprising the genes,transformants comprising the vectors, an antibodies against theproteins; and therapeutic agents for nervous diseases.

In one embodiment, the present invention relates to a recombinantprotein having a protein comprising at least a portion of the amino acidsequence set forth in SEQ ID NO:2. In some embodiments, the presentinvention provides a protein which consists of the amino acid sequenceshown in SEQ ID NO: 2 having a deletion, substitution, or addition of atleast one amino acid and which has neurogenesis inducing activity. Inanother embodiment, the present invention relates to a neurogenesisinducing gene encoding for a protein comprising at least a portion ofthe amino acid sequence set forth in SEQ ID NO:2 or a neurogenesisinducing gene encoding for a protein comprising at least a portion ofthe amino acid sequence set forth in SEQ ID NO:2 having a deletion,substitution, or addition of at least one amino acid and which hasneurogenesis inducing activity. The present invention also relates to agene which hybridizes with the neurogenesis inducing gene encoding for aprotein comprising at least a portion of the amino acid sequence setforth in SEQ ID NO:2.

In another embodiment, the present invention relates to a genecomprising a DNA sequence having at least a portion of the nucleotidesequence set forth in SEQ ID NO: 1. The present invention also relatesto a DNA which hybridizes with a DNA having at least a portion of thenucleotide sequence set forth in SEQ ID NO:1, and which codes for aprotein having neurogenesis inducing activity.

In another embodiment, the present invention relates to an isolatednucleic acid encoding a protein having at least a portion of the aminoacid sequence selected from the group consisting of SEQ ID NO: 2, SEQ IDNO: 42 and in SEQ ID NO: 44. In one embodiment, the nucleic acidcomprises a nucleotide sequence having at least a portion of thesequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:41, and SEQ ID NO: 43. In preferred embodiments, the present inventionprovides a nucleic acid encoding a neurogenesis inducing gene (e.g.,early neurogenesis inducing gene). In particular, the present inventionprovides a nucleic acid encoding a gene selected from the groupconsisting of Zic1, Zic 2, and Zic3 genes. In one embodiment, the geneis a Xenopus gene.

The present invention further relates to a nucleic acid capable ofhybridizing to a nucleic acid comprising a nucleotide sequence having atleast a portion of the sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO: 41, and SEQ ID NO: 43. In a preferredembodiment, the nucleic acid capable of hybridizing under stringentconditions.

In yet another embodiment, the present invention also relates torecombinant vectors comprising at least a portion of genes describedabove. The present invention further relates to transformants (e.g., ahost cell) comprising the recombinant vectors of the present invention.

The present invention also relates to compositions comprising a proteinhaving at least a portion of the amino acid sequence selected from thegroup consisting of SEQ ID NO: 2, SEQ ID NO: 42, and SEQ ID NO: 44. Inone embodiment, the protein is selected from the group consisting ofZic1, Zic2, and Zic3 proteins. In another embodiment, the protein is aXenopus protein. In yet another embodiment, the protein is a recombinantprotein.

The present invention also provides antibodies against the abovedescribed proteins. In particular, the present invention relates to apurified antibody specific to a protein having at least a portion of theamino acid sequence selected from the group consisting of SEQ ID NO: 2,SEQ ID NO: 42, and SEQ ID NO: 44. The present invention further providestherapeutic agents for nervous diseases associated with the aboveproteins (e.g., in the presence or absence of the proteins). Inpreferred embodiments, the present invention relates to therapeuticagents or agents for gene therapy of nervous diseases including, but notlimited to Alzheimer's disease, amyotrophic lateral sclerosis,spinocerebellar degeneration, Parkinson's disease and cerebral ischemia,although it is not intended that the present invention be limited totherapeutic agents related to these diseases.

Furthermore, the present invention relates to methods for producing aneurogenesis inducing protein, comprising, for example, the steps ofculturing a transformant comprising a recombinant vector discussed aboveand recovering the neurogenesis inducing protein from the resultantculture. In one embodiment, the methods of the present inventioncomprises the steps of: a) providing a composition comprising arecombinant vector, wherein the recombinant vector comprises a nucleicacid having at least a portion of the sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 41 and SEQ ID NO: 43, and a hostcell; b) transforming the recombinant vector into the host cell toproduce a transformant; and c) culturing the transformant to produce aneurogenesis inducing protein. In one embodiment, the methods of thepresent invention further comprises the step of d) isolating theneurogenesis inducing protein. In preferred embodiments, theneurogenesis inducing protein is an early neurogenesis inducing protein.

Further, the present invention relates to methods for gene therapy,comprising the steps of: a) providing i) a subject, and ii) acomposition comprising a nucleic acid having at least a portion of thesequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:41 and SEQ ID NO: 43; and b) delivery the composition to the subject. Inpreferred embodiments, the subject suffers from a disease selected fromthe group consisting of Alzheimer's disease, amyotrophic lateralsclerosis, spinocerebellar degeneration, Parkinson's disease andcerebral ischemia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents photographs showing the results of expression tests ofthe Zic3 gene of the invention (morphology of an organism).

FIG. 2 presents autoradiographs showing the results of expression testsof the Zic3 gene.

FIG. 3 presents autoradiographs showing the results of expression testsof the Zic3 gene.

FIG. 4 presents autoradiographs showing the results of expression testsof the Zic3 gene and photographs showing morphology of an organism.

FIG. 5 presents photographs showing the results of expression tests ofthe Zic3 gene (morphology of an organism).

FIG. 6 presents photographs showing the results of expression tests ofthe Zic3 gene (morphology of an organism).

FIG. 7 presents autoradiographs showing the results of expression testsof the Zic3 gene.

FIG. 8 shows the sequences of Xenopus Zic1 and Zic2. Panel A and Panel Bprovide the predicted amino acid sequences of Xenopus Zic1 (SEQ IDNO:42) and Zic2 (SEQ ID NO:44), respectively. In both panels, theunderlining indicates the location of the zinc finger motif. Panel Cshows the alignment of the zinc finger domain of Xenopus Zic1 (SEQ IDNO:50), Zic2 (SEQ ID NO:51), Zic3 (SEQ ID NO:52; and Nakata et at.,“Xenopus Zic3, a primary regulator both in neural and neural crestdevelopment,” Proc. Natl. Acad. Sci. USA 94:11980-11985 [1997]), mouseZic1 (SEQ ID NO:53; and Aruga et at., “A novel zinc finger protein, Zic,is involved in neurogenesis, especially in the cell lineage ofcerebellar granule cells,” J. Neurochem. 63: 1880-1890 [1994]), Zic2(SEQ ID NO:54), Zic3 (SEQ ID NO:55) and Zic 3 (SEQ ID NO:56; Aruga etal., “The mouse Zic gene family Homologues of the Drosophila pair-rulegene odd-paired,” J. Biol. Chem. 271: 1043-1047 [1996]; Aruga et al.,“Identification and characterization of Zic4, a new member of the mouseZic gene family,” Gene 172: 291-294 [1996]) and Opa (SEQ ID NO:57; andBenedyk et al., “Odd-paired: a zinc finger pair-rule protein requiredfor the timely activation of engrailed and wingless in Drosophilaembryos,” Genes. Dev. 8: 105-117 [1994]). The asterisks indicate theamino acids conserved among all eight proteins. The bold letters andasterisks indicate the cysteine and histidine residues of the C2H2motif. Panel D shows a conserved portion of the N-terminal region amongZic family (SEQ ID NOS:58-63) and Opa (SEQ ID NO:64).

FIG. 9 shows the temporal expression profiles of Xenopus Zic1, Zic2 andZic3 during Xenopus development. RNA was extracted from embryos at theindicated stage of development and Zic1, Zic2, and Zic3 mRNA expressionlevels were measured by RT-PCR. The ubiquitous marker Histone H4 servedas a control.

FIG. 10 shows the spatial expression patterns of Zic1 and Zic2 inXenopus embryos. Series of embryos were hybridized withdigoxigenin-labeled antisense Zic1 (A-L) and Zic2 (M-X) RNA (A, m)blastula stage (stage 9). In the blastula stage, Zic1 and Zic2 wereexpressed throughout the ectoderm (A, animal side; V, vegetal side)(B,C,N,O) mid-gastrula stage (stage 10.5). (C) The expression of Zic1 inthe prospective neural plate was confirmed in a cross-section of thesame embryo as in B (arrowhead). However, Zic2 was expressed in abroader region of the ectoderm (N,O). (D,P) Stage 11-12 (E,Q) Stage12.5-13.5 (F,R) Stage 14-15. White and black arrowheads indicate thelateral edge of the neural plate and the neural crest, respectively.Arrows indicate the neural plate border region of the prospectiverhombencephalon (G,H,S,T) Stage 20. The black arrowhead, white arrowheadand arrow indicate the telencephalon, diencephalon and mesencephalon,respectively. (I-L,U-X) Stage 30. Transverse section through the head(J,V) and trunk region (L,X) of the same embryo as in (I,U). (K,W)magnified views in the head legion of I and U. (A,M,I,U) are lateralviews. The upper side of the panel is the animal side in (A,M). (B,N)Dorsal-vegetal views, (D-G,P-S) Dorsal views. (D-F,P-R) anterior side isupper side. (G,S) The anterior side is toward the left. (H,T) Anteriorviews of (G,S). The upper side is dorsal (dl), dorsal lip; blastopore(bp); somite (s.); neural tube (nt); optic vesicle (ov); telencephalon(tel); diencephalon and midbrain boundary (di/mi); rhombencephalon (rh);eye (e); ear vesicle (ev); cement gland (cg).

FIG. 11 shows Zic1 or Zic2 overexpression induces ectopic pigment cellexpression in embryo, as does overexpression of Zic3. A total of 100 or500 pg of MT-Zic3 (B), MT-Zic1 (C, D), or MT-Zic2 (E, F) mRNA wasinjected into two blastomeres of 2-cell stage embryos, which were thencultured to stage 28-29 (A) is uninjected. Clusters of pigment cellsappeared in MT-Zic3, MT-Zic1 or MT-Zic2 mRNA-injected embryos (B,D,E,F,arrowheads). Transverse sections of MT-Zic1 (G) or MT-Zic2 (H)mRNA-injected embryos. A total of 250 pg of MT-Zic1 (G) or 125 pg ofMT-Zic2 (H) mRNA was injected into one blastomere of 2-cell stageembryos and embryos were cultured to stage 35-36. Ectopic expression ofpresumptive mesenchymal tissues was observed in the MT-Zic1 or MT-Zic2mRNA-injected side (G,H, arrow-heads). (I) Western blot analysis of eachZic protein level in MT-Zic3, MT-Zic1 or MT-Zic2 mRNA-injected embryos.A total of 100 pg of MT-Zic3, MT-Zic1 or MT-Zic2 mRNA was injected intotwo blastomeres of 2-cell stage embryos and cultured to stage 10.5. Atotal of 10 pg of protein prepared from whole embryo was analyzed byWestern blot analysis using an anti-myc antibody.

FIG. 12 shows the pigment cells expressed by Zic1 or Zic2 overexpressionin animal cap explant. (A) Uninjected animal cap explant. A total of 250pg of MT-Zic1 (B) or a total of 125 pg MT-Zic2 (C) mRNA was injectedinto two animal blastomeres of 2-cell stage embryos obtained by themating between albino female and wild type males. Animal caps wereexplanted at stage 9 and cultured. (D) A magnified view of the abdominalregion of the control embryo at the same time as animal cap explant(stage 45). The pigment cells appeared in the MT-Zic1 or MT-Zic2overexpressed animal cap explants are similar to the melanocytes whichare derived from neural crest.

FIG. 13 shows that Zic1 and Zic2 induced NCAM and Xslu expression butreduced epidermis in early stage embryos. A total of 250 pg of MT-Zic1mRNA (A-C) or 125 pg of MT-Zic2 (D-F) was injected into one blastomereof 2-cell stage embryos. In situ hybridization was performed with apan-neural marker gene (NCAM; A,D), a neural crest marker (Xslu; B,E)probe, and immunohistochemistry was performed with an epidermal marker(EpA) monoclonal antibody (C,F). Dorsal view of a stage 13-14 embryo.NCAM and Xslu expressing regions show lateral expansion of the injectedside (A,B,D,E). EpA staining in the epidermis is reduced on the injectedside (C,F).

FIG. 14 shows that Zic1 and Zic2-induced anterior neural marker genesand a neural crest marker gene without mesoderm induction in animal capexplants, as seen with Zic3. Embryos were injected with MT-Zic3 orMT-Zic1 mRNA at the two-cell stage. Animal caps were explanted at stage9 and cultured. When sibling embryos reached stage 25, the expression ofanterior-posterior marker genes (En2 and HoxB9 [(=Xlhbox6]), apan-neural marker gene (NCAM), a neuronal differentiation marker(N-tubulin), a neural crest marker (Xtwi), and a dorsal mesodermalmarker (M. actin; muscle actin) were examined by RT-PCR. Althoughuninjected (Uninj.) animal caps expressed none of these markers, animalcaps injected with Zic1, Zic2, or Zic3 mRNA expressed the anteriormarker (En2) and the neural crest marker (Xtwi) while expressing neitherthe posterior marker (HOXB9) nor the dorsal mesodermal marker (M.actin).In each experiment, sibling control embryos served as a positive control(Embryo) and PCR on the same RNA without reverse transcription was doneto verify the absence of genomic DNA (RT-PCR)

GENERAL DESCRIPTION OF THE INVENTION

The present invention relates to genes capable of inducing neurogenesis(e.g., early neurogenesis). In particular, the present invention relatesto Zic1, Zic2, and Zic 3 genes, vectors containing such genes, hostcells containing such vectors, proteins produced by such host cells,antibodies raised to such proteins, and therapeutic agents or agents forgene therapy of nervous diseases.

The Zic family was originally identified as a group of genes encodingzinc finger proteins expressed in adult mouse cerebella (Aruga et al.,J. Neurochem 63: 1880-1890 [1994], supra). In mice, at least four kindsof Zic genes have been identified (Aruga et al., J. Neurochem 63:1880-1890 [1994], supra; Aruga et al., J. Biol. Chem. 271: 1043-1047[1996], supra; Aruga et al., Gene 172: 291-294 [1996], supra). Thesezinc finger proteins share a highly conserved domain consisting of fivetandemly repeated C₂H₂-type zinc finger motifs. The motifs are highlyconserved among various species (Benedyk et al., supra; Cimbora andSakonju, “Drosophila midgut morphogenesis requires the function of thesegmentation gene odd-paired,” Dev. Biol. 169: 580-595 [1995]; Yokota etal., “Predominant expression of human Zic in cerebellar granule celllineage and medulloblastoma,” Cancer Res. 56: 377-383 [1996]; Gebbia etal., “X-linked situs abnormalities result from mutations in ZIC3,”Nature Genet. 17: 305-308 [1997]; Nakata et al., supra), including theirDrosophila homologue odd-paired, which plays important roles inparasegmental subdivision and visceral mesoderm development of theDrosophilia embryo.

The mouse Zic1, Zic 2, and Zic3 genes are expressed in a similar butdistinct manner during gestational development (Nagai et al., “Theexpression of the mouse Zic1, Zic2 and Zic3 gene suggests an essentialrole for Zic genes in body pattern formation,” Dev. Biol. 182: 299-313[1997]). The expression of these genes was detectable at the primitivestreak stage and later in neural tissue, somites and limb buds. Althoughthey are expressed in overlapping sites, their respective expressionpatterns are not identical. These findings suggest that each Zic genehas specific roles in vertebrate development. This has been confirmed infunctional studies. For example, the disruption of mouse Zic1 generesults in malformation of the central nervous system, particularly, thecerebellum (Aruga et al., “Mouse Zic1 is involved in cerebellardevelopment,” J. Neurosci. 18: 284-293 [1998]), whereas a mutation inhuman Zic3 results in disturbance of the left to right body axis (Gebbiaet al., supra). The present invention contemplates that Zic genesconstitute a multigene family in other vertebrates, and that theirrespective roles are not identical, although an understanding of themechanism is not necessary in order to practice the present invention,nor is it intended that the present invention be so limited.

In some embodiments, the present invention relates to the role ofXenopus Zic genes in early vertebrate development. Xenopus Zic3 gene hasbeen cloned (Nakata et al., supra). Expression of Xenopus Zic3 isdetected in the prospective neural plate region at gastrulation. Theonset of its expression is earlier than those of most proneural genesand follows chordin expression. Zic3 expression is induced by blockadeof the BMP4 signal. Overexpression of Zic3 results in hyperplasticneural and neural crest-derived tissue. In an animal cap explant,overexpression of Zic3 induces expression of proneural genes such asNeurogenin (Xngnr-1) and neural crest genes. These findings show thatXenopus Zic3 can determine ectodermal cell fate and promote the earlieststep of neural and neural crest development.

Experiments conducted during the development of the present inventionfurther identified and characterized Xenopus Zic-related genes, Zic1 andZic2, and examined their expression patterns and functions. At leastthree Zic genes exist in Xenopus laevis, and their respective homologuesin mice had been reported. The homologues of Zic1, Zic2 and Zic3 havealso been confirmed in some other vertebrates (See e.g., Yokota et al.,supra; and Gebbia et al., supra), suggesting that Zic1, Zic2 and Zic3 isimportant in the vertebrate development.

The present invention has determined the expression patterns of thethree Zic genes in Xenopus embryos (Table 1) (See also, Nakata et al.,supra). Table 1 shows several similarities and differences among thethree Zic genes. First of all, Zic2 was maternally expressed. Thedefinitive roles of Zic2 during this period remains unclear at thistime. Although an understanding of the mechanisms is not necessary forthe practice of the present invention and the present invention is notlimited to any particular mechanism, post-transcriptional regulatorymechanisms may work. Some other genes involved in early neuraldevelopment have also been detected as maternal messages (e.g., Otx2,Neurogenin; See also, Pannese et al., “The Xenopus homologue of Otx2 isa maternal homeobox gene that demarcates and specifies anterior bodyregions,” Development 121: 707-720 [1995]). The role of Zic1 and Zic2before zygotic transcription is clarified further below.

The three Xenopus Zic genes have similar expression patterns in neuraltissue. All three genes are expressed in the prospective neural plateregion at the time of neural induction. Expression in the medial part ofthe neural plate was diminished while that in the neural plate borderregion increased. Thereafter, expression increased on the anterior anddorsal side with regard to the anterior-posterior and dorsal-ventralaxes, respectively. These patterns of expression show that Zic genesplay a role in neural induction and the patterning of neural tissues inthe early phase of neural development.

TABLE 1 Comparative summary of the expression of the three Zic genes:Stage Tissues Zic1 Zic2 Zic3   9 (blastula) Ectoderm +++ +++ −   10.5(gastrula) Prospective neural plate ++ ++ +++ Prospective epidermis − +− 14-16 (neurula) Neural fold +++ +++ +++   20 (early tailbud) Dorsalbrain ++ ++ ++ Dorsal spinal cord ++ ++ + Eye vesicle − +++ − Somite +++++ −   30 (tailbud) Telencephaon ++ + +++ Diencephalon ++ + +++Mesencephalon ++ + ++ Rhombencephalon ++ + ++ Dorsal spinal cord ++ ++ +Eye (ciliary marginal zone) − +++ − Somite +++ ++ − Lateral mesoderm(tail) − − ++ Cement gland − + − Posterior ventral epidermis − + −

The differences in spatial Zic gene expression in neural tissues wereprincipally different levels of expression along the anterior-posterioraxis and expression in the eye (see Table 1). In addition to theexpression in neural tissues, that in somites and their derivativesshowed variability. In particular, Xenopus Zic1 is strongly expressed inthe somites. It was found that mouse Zic1 is similarly expressed in thesomites and plays a critical role in the development of somitederivatives (Nagai et al., supra), suggesting that the Xenopus Zic1 mayalso play a role in somite development.

The expression in somites is well correlated between Xenopus and mouseZic genes in that the expression is high for Zic1, moderate for Zic2 andvery low for Zic3 both in Xenopus and mouse. As a consequence, theexpression patterns of Xenopus Zic1, Zic2, and Zic3 correspond to thoseof mouse Zic1, Zic2, and Zic3, respectively. Thus, the present inventioncontemplates that the roles of Zic1, Zic2, and Zic3 are generally wellconserved between Xenopus and mouse.

Xenopus Zic1 and Zic2 are novel neuralizing factors. Experimentsconducted during the development of the present invention showed thatXenopus Zic1 and Zic2 were capable of inducing neural and neural cresttissues. Zic1 or Zic2 overexpression in embryos resulted in theenlargement of neural plates and neural plate border regions in neurulaand the appearance of ectopic pigment cells which were derived from theneural crest. Furthermore, overexpression leads to the induction ofneural and neural crest markers in the animal cap explants. Thus, thepresent invention provides compositions that serve as regulators ofneural and neural crest development.

The present invention demonstrates that Xenopus Zic3 is a primaryregulator of neural and neural crest development (See also, Nakata etal., supra). The present invention also shows that Zic1 or Zic2overexpression yielded essentially the same results as observed withZic3 overexpression. Ectopic pigment cells in embryos overexpressingZic2 were equivalent to those found in Zic3-overexpressing embryos.However, the induced ectopic pigment cells were less dense in theZic1-overexpressed embryos than in the Zic2 or Zic3-overexpressedembryos (FIG. 11). In addition, a larger amount of RNA was required toinduce neural and neural crest markers in animal cap explants to thesame extent (FIG. 14). Although an understanding of the mechanisms isnot necessary for the practice of the present invention and the presentinvention is not limited to any particular mechanism, these resultsindicate that the potency of neural and neural crest induction by Zic1is less than that of Zic2 and Zic3. This finding suggests that Zic1 mayplay a supportive role in the Zic2 and Zic3-mediated neural and neuralcrust induction. This situation is analogous to the relationship betweenmouse En1 and En2, in which the En1 and En2 proteins play the same rolesin midbrain and hindbrain development (Hanks et al., “Rescue of the En-1mutant phenotype by replacement of En-1 with En-2,” Science 269, 679-682[995]).

Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the term “early neurogenesis” refers to the process ofneurogenesis related to the formation of the neural plate from theectoderm (e.g., from the late blastula stage to the neurula stage ofXenopus embryos). The term “early neurogenesis inducing activity” refersto an activity which gives rise to the neural plate and other tissues(e.g., the neural crest) from the ectoderm. The term “neurogenesis”refers to the process of neurogenesis as a whole, including thedevelopment, differentiation, and maturation of the nervous system afterthe early neurogenesis.

The term “agonist,” as used herein, refers to a molecule which, wheninteracting with an biologically active molecule, causes a change (e.g.,enhancement) in the biologically active molecule, which modulates theactivity of the biologically active molecule. Agonists may includeproteins, nucleic acids, carbohydrates, or any other molecules whichbind or interact with biologically active molecules. For example,agonists can alter the activity of gene transcription by interactingwith RNA polymerase directly or through a transcription factor.

The terms “antagonist” or “inhibitor,” as used herein, refer to amolecule which, when interacting with a biologically active molecule,blocks or modulates the biological activity of the biologically activemolecule. Antagonists and inhibitors may include proteins, nucleicacids, carbohydrates, or any other molecules that bind or interact withbiologically active molecules. Inhibitors and antagonists can effect thebiology of entire cells, organs, or organisms (e.g., an inhibitor thatslows neuron degeneration).

The term “modulate,” as used herein, refers to a change in thebiological activity of a biologically active molecule. Modulation can bean increase or a decrease in activity, a change in bindingcharacteristics, or any other change in the biological, functional, orimmunological properties of biologically active molecules.

As used herein, the term “nucleic acid molecule” refers to any nucleicacid containing molecule including but not limited to DNA or RNA. Theterm encompasses sequences that include any of the known base analogs ofDNA and RNA including, but not limited to, 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of a polypeptideor precursor (e.g., Zic1, Zic2, and Zic3). The polypeptide can beencoded by a full length coding sequence or by any portion of the codingsequence so long as the desired activity or functional properties (e.g.,enzymatic activity, ligand binding, signal transduction, etc.) of thefull-length or fragment are retained. The term also encompasses thecoding region of a structural gene and the including sequences locatedadjacent to the coding region on both the 5′ and 3′ ends for a distanceof about 1 kb or more on either end such that the gene corresponds tothe length of the full-length mRNA. The sequences that are located 5′ ofthe coding region and which are present on the mRNA are referred to as5′ non-translated sequences. The sequences that are located 3′ ordownstream of the coding region and which are present on the mRNA arereferred to as 3′ non-translated sequences. The term “gene” encompassesboth cDNA and genomic forms of a gene. A genomic form or clone of a genecontains the coding region interrupted with non-coding sequences termed“introns” or “intervening regions” or “intervening sequences.” Intronsare segments of a gene which are transcribed into nuclear RNA (hnRNA);introns may contain regulatory elements such as enhancers. Introns areremoved or “spliced out” from the nuclear or primary transcript; intronstherefore are absent in the messenger RNA (mRNA) transcript. The mRNAfunctions during translation to specify the sequence or order of aminoacids in a nascent polypeptide.

As used herein, the term “gene expression” refers to the process ofconverting genetic information encoded in a gene into RNA (e.g., mRNA,rRNA, tRNA, or snRNA) through “transcription” of the gene (i.e., via theenzymatic action of an RNA polymerase), and for protein encoding genes,into protein through “translation” of mRNA. Gene expression can beregulated at many stages in the process. “Up-regulation” or “activation”refers to regulation that increases the production of gene expressionproducts (i.e., RNA or protein), while “down-regulation” or “repression”refers to regulation that decrease production. Molecules (e.g.,transcription factors) that are involved in up-regulation ordown-regulation are often called “activators” and “repressors,”respectively.

Where “amino acid sequence” is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, “amino acidsequence” and like terms, such as “polypeptide” or “protein” are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequenceswhich are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers that control or influence thetranscription of the gene. The 3′ flanking region may contain sequenceswhich direct the termination of transcription, post-transcriptionalcleavage and polyadenylation.

The term “wild-type” refers to a gene or gene product which has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the term“modified” or “mutant” refers to a gene or gene product which displaysmodifications in sequence and or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product. Itis noted that naturally-occurring mutants can be isolated; these areidentified by the fact that they have altered characteristics whencompared to the wild-type gene or gene product.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

DNA molecules are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides or polynucleotidesin a manner such that the 5′ phosphate of one mononucleotide pentosering is attached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage. Therefore, an end of an oligonucleotides orpolynucleotide, referred to as the “5′ end” if its 5′ phosphate is notlinked to the 3′ oxygen of a mononucleotide pentose ring and as the “3′end” if its 3′ oxygen is not linked to a 5′ phosphate of a subsequentmononucleotide pentose ring. As used herein, a nucleic acid sequence,even if internal to a larger oligonucleotide or polynucleotide, also maybe said to have 5′ and 3′ ends. In either a linear or circular DNAmolecule, discrete elements are referred to as being “upstream” or 5′ ofthe “downstream” or 3′ elements. This terminology reflects the fact thattranscription proceeds in a 5′ to 3′ fashion along the DNA strand. Thepromoter and enhancer elements that direct transcription of a linkedgene are generally located 5′ or upstream of the coding region. However,enhancer elements can exert their effect even when located 3′ of thepromoter element or the coding region. Transcription termination andpolyadenylation signals are located 3′ or downstream of the codingregion.

As used herein, the terms “an oligonucleotide having a nucleotidesequence encoding a gene” and “polynucleotide having a nucleotidesequence encoding a gene,” means a nucleic acid sequence comprising thecoding region of a gene or in other words the nucleic acid sequencewhich encodes a gene product. The coding region may be present in eithera cDNA, genomic DNA or RNA form. When present in a DNA form, theoligonucleotide or polynucleotide may be single-stranded (ie., the sensestrand) or double-stranded. Suitable control elements such asenhancers/promoters, splice junctions, polyadenylation signals, etc. maybe placed in close proximity to the coding region of the gene if neededto permit proper initiation of transcription and/or correct processingof the primary RNA transcript. Alternatively, the coding region utilizedin the expression vectors of the present invention may containendogenous enhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. or a combination of both endogenous andexogenous control elements.

As used herein, the term “oligonucleotide,” refers to a short length ofsingle-stranded polynucleotide chain. Oligonucleotides are typicallyless than 100 residues long (e.g., between 15 and 50), however, as usedherein, the term is also intended to encompass longer polynucleotidechains. Oligonucleotides are often referred to by their length. Forexample a 24 residue oligonucleotide is referred to as a “24-mer”.Oligonucleotides can form secondary and tertiary structures byself-hybridizing or by hybridizing to other polynucleotides. Suchstructures can include, but are not limited to, duplexes, hairpins,cruciforms, bends, and triplexes.

As used herein, the term “regulatory element” refers to a geneticelement which controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element thatfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements are splicing signals, polyadenylationsignals, termination signals, etc. (defined infra).

Transcriptional control signals in eukaryotes comprise “promoter” and“enhancer” elements. Promoters and enhancers consist of short arrays ofDNA sequences that interact specifically with cellular proteins involvedin transcription (T. Maniatis et al., Science 236:1237 [1987]). Promoterand enhancer elements have been isolated from a variety of eukaryoticsources including genes in yeast, insect and mammalian cells, andviruses (analogous control elements, i.e., promoters, are also found inprokaryote). The selection of a particular promoter and enhancer dependson what cell type is to be used to express the protein of interest. Someeukaryotic promoters and enhancers have a broad host range while othersare functional in a limited subset of cell types (for review see, S. D.Voss et al., Trends Biochem. Sci., 11:287 [1986]; and T. Maniatis etal., supra). For example, the SV40 early gene enhancer is very active ina wide variety of cell types from many mammalian species and has beenwidely used for the expression of proteins in mammalian cells (R.Dijkema et al., EMBO J. 4:761 [1985]). Two other examples ofpromoter/enhancer elements active in a broad range of mammalian celltypes are those from the human elongation factor 1α gene (T. Uetsuki etal., J. Biol. Chem., 264:5791 [1989]; D. W. Kim et al., Gene 91:217[1990]; and S. Mizushima and S. Nagata, Nuc. Acids. Res., 18:5322[1990]) and the long terminal repeats of the Rous sarcoma virus (C. M.Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777 [1982]) and the humancytomegalovirus (M. Boshart et al., Cell 41:521 [1985]). Some promoterelements serve to direct gene expression in a tissue-specific manner.

As used herein, the term “promoter/enhancer” denotes a segment of DNAwhich contains sequences capable of providing both promoter and enhancerfunctions (i.e., the functions provided by a promoter element and anenhancer element, see above for a discussion of these functions). Forexample, the long terminal repeats of retroviruses contain both promoterand enhancer functions. The enhancer/promoter may be “endogenous” or“exogenous” or “heterologous.” An “endogenous” enhancer/promoter is onewhich is naturally linked with a given gene in the genome. An“exogenous” or “heterologous” enhancer/promoter is one which is placedin juxtaposition to a gene by means of genetic manipulation (i.e.,molecular biological techniques such as cloning and recombination) suchthat transcription of that gene is directed by the linkedenhancer/promoter.

The presence of “splicing signals” on an expression vector often resultsin higher levels of expression of the recombinant transcript. Splicingsignals mediate the removal of introns from the primary RNA transcriptand consist of a splice donor and acceptor site (J. Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, New York [1989], pp. 16.7-16.8). A commonly usedsplice donor and acceptor site is the splice junction from the 16S RNAof SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cellsrequires expression of signals directing the efficient termination andpolyadenylation of the resulting transcript. Transcription terminationsignals are generally found downstream of the polyadenylation signal andare a few hundred nucleotides in length. The term “poly A site” or “polyA sequence” as used herein denotes a DNA sequence that directs both thetermination and polyadenylation of the nascent RNA transcript. Efficientpolyadenylation of the recombinant transcript is desirable astranscripts lacking a poly A tail are unstable and are rapidly degraded.The poly A signal utilized in an expression vector may be “heterologous”or “endogenous.” An endogenous poly A signal is one that is foundnaturally at the 3′ end of the coding region of a given gene in thegenome. A heterologous poly A signal is one that is isolated from onegene and placed 3′ of another gene. A commonly used heterologous poly Asignal is the SV40 poly A signal. The SV40 poly A signal is contained ona 237 bp BamHI/BclI restriction fragment and directs both terminationand polyadenylation (J. Sambrook, supra, at 16.6-16.7).

Eukaryotic expression vectors may also contain “viral replicons” or“viral origins of replication.” Viral replicons are viral DNA sequencesthat allow for the extrachromosomal replication of a vector in a hostcell expressing the appropriate replication factors. Vectors thatcontain either the SV40 or polyoma virus origin of replication replicateto high “copy number” (up to 10⁴ copies/cell) in cells that express theappropriate viral T antigen. Vectors that contain the replicons frombovine papillomavirus or Epstein-Barr virus replicate extrachromosomallyat “low copy number” (˜100 copies/cell).

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, for the sequence“A-G-T,” is complementary to the sequence “T-C-A.” Complementarity maybe “partial,” in which only some of the nucleic acids' bases are matchedaccording to the base pairing rules. Also, there may be “complete” or“total” complementarity between the nucleic acids. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions, aswell as detection methods that depend upon binding between nucleicacids.

The term “homology” refers to a degree of complementarity. There may bepartial homology or complete homology (i.e., identity). A partiallycomplementary sequence is one that at least partially inhibits acompletely complementary sequence from hybridizing to a target nucleicacid is referred to using the functional term “substantiallyhomologous.” The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or Northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or probe will compete for and inhibit the binding(i.e., the hybridization) of a completely homologous to a target underconditions of low stringency. This is not to say that conditions of lowstringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second target thatlacks even a partial degree of complementarity (e.g., less than about30% identity); in the absence of non-specific binding the probe will nothybridize to the second non-complementary target.

The art knows well that numerous equivalent conditions may be employedto comprise low stringency conditions; factors such as the length andnature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,etc.) and the concentration of the salts and other components (e.g., thepresence or absence of formamide, dextran sulfate, polyethylene glycol)are considered and the hybridization solution may be varied to generateconditions of low stringency hybridization different from, butequivalent to, the above listed conditions. In addition, the art knowsconditions that promote hybridization under conditions of highstringency (e.g., increasing the temperature of the hybridization and/orwash steps, the use of formamide in the hybridization solution, etc.)(see definition below for “stringency”).

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described above.

A gene may produce multiple RNA species that are generated bydifferential splicing of the primary RNA transcript. cDNAs that aresplice variants of the same gene will contain regions of sequenceidentity or complete homology (representing the presence of the sameexon or portion of the same exon on both cDNAs) and regions of completenon-identity (for example, representing the presence of exon “A” on cDNA1 wherein cDNA 2 contains exon “B” instead). Because the two cDNAscontain regions of sequence identity they will both hybridize to a probederived from the entire gene or portions of the gene containingsequences found on both cDNAs; the two splice variants are thereforesubstantially homologous to such a probe and to each other.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe that can hybridize(i.e., it is the complement of) the single-stranded nucleic acidsequence under conditions of low stringency as described above.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree of thecomplementary between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids. A single molecule that contains pairing of complementarynucleic acids within its structure is said to be “self-hybridized.”

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the T_(m)of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (See e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization [1985]). Other referencesinclude more sophisticated computations that take structural as well assequence characteristics into account for the calculation of T_(m).

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. With “high stringency” conditions, nucleicacid base pairing will occur only between nucleic acid fragments thathave a high frequency of complementary base sequences. Thus, conditionsof “weak” or “low” stringency are often required with nucleic acids thatare derived from organisms that are genetically diverse, as thefrequency of complementary sequences is usually less.

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

Template specificity is achieved in most amplification techniques by thechoice of enzyme. Amplification enzymes are enzymes that, underconditions they are used, will process only specific sequences ofnucleic acid in a heterogeneous mixture of nucleic acid. For example, inthe case of Qβ replicase, MDV-1 RNA is the specific template for thereplicase (D. L. Kacian et al., Proc. Natl. Acad. Sci. USA 69:3038[1972]). Other nucleic acid will not be replicated by this amplificationenzyme. Similarly, in the case of T7 RNA polymerase, this amplificationenzyme has a stringent specificity for its own promoters (M. Chamberlinet al., Nature 228:227 [1970]). In the case of T4 DNA ligase, the enzymewill not ligate the two oligonucleotides or polynucleotides, where thereis a mismatch between the oligonucleotide or polynucleotide substrateand the template at the ligation junction (D. Y. Wu and R. B. Wallace,Genomics 4:560 [1989]). Finally, Taq and Pfu polymerases, by virtue oftheir ability to function at high temperature, are found to display highspecificity for the sequences bounded and thus defined by the primers;the high temperature results in thermodynamic conditions that favorprimer hybridization with the target sequences and not hybridizationwith non-target sequences (H. A. Erlich (ed.), PCR Technology, StocktonPress [1989]).

As used herein, the term “amplifiable nucleic acid” is used in referenceto nucleic acids which may be amplified by any amplification method. Itis contemplated that “amplifiable nucleic acid” will usually comprise“sample template.”

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample that is analyzed for the presence of “target”.In contrast, “background template” is used in reference to nucleic acidother than sample template which may or may not be present in a sample.Background template is most often inadvertent. It may be the result ofcarryover, or it may be due to the presence of nucleic acid contaminantssought to be purified away from the sample. For example, nucleic acidsfrom organisms other than those to be detected may be present asbackground in a test sample.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, that is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product that is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, that is capable of hybridizing to another oligonucleotideof interest. A probe may be single-stranded or double-stranded. Probesare useful in the detection, identification and isolation of particulargene sequences. It is contemplated that any probe used in the presentinvention will be labelled with any “reporter molecule,” so that isdetectable in any detection system, including, but not limited to enzyme(e.g., ELISA, as well as enzyme-based histochemical assays),fluorescent, radioactive, and luminescent systems. It is not intendedthat the present invention be limited to any particular detection systemor label.

As used herein, the term “target,” refers to the region of nucleic acidbounded by the primers. Thus, the “target” is sought to be sorted outfrom other nucleic acid sequences. A “segment” is defined as a region ofnucleic acid within the target sequence.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe method of K. B. Mullis U.S. Pat. Nos. 4,683,195 4,683,202, and4,965,188, hereby incorporated by reference, which describe a method forincreasing the concentration of a segment of a target sequence in amixture of genomic DNA without cloning or purification. This process foramplifying the target sequence consists of introducing a large excess oftwo oligonucleotide primers to the DNA mixture containing the desiredtarget sequence, followed by a precise sequence of thermal cycling inthe presence of a DNA polymerase. The two primers are complementary totheir respective strands of the double stranded target sequence. Toeffect amplification, the mixture is denatured and the primers thenannealed to their complementary sequences within the target molecule.Following annealing, the primers are extended with a polymerase so as toform a new pair of complementary strands. The steps of denaturation,primer annealing and polymerase extension can be repeated many times(i.e., denaturation, annealing and extension constitute one “cycle”;there can be numerous “cycles”) to obtain a high concentration of anamplified segment of the desired target sequence. The length of theamplified segment of the desired target sequence is determined by therelative positions of the primers with respect to each other, andtherefore, this length is a controllable parameter. By virtue of therepeating aspect of the process, the method is referred to as the“polymerase chain reaction” (hereinafter “PCR”). Because the desiredamplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified”.

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g., hybridization with a labeled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;incorporation of ³²P-labeled deoxynucleotide triphosphates, such as dCTPor dATP, into the amplified segment). In addition to genomic DNA, anyoligonucleotide or polynucleotide sequence can be amplified with theappropriate set of primer molecules. In particular, the amplifiedsegments created by the PCR process are, themselves, efficient templatesfor subsequent PCR amplifications.

As used herein, the terms “PCR product,” “PCR fragment,” and“amplification product” refer to the resultant mixture of compoundsafter two or more cycles of the PCR steps of denaturation, annealing andextension are complete. These terms encompass the case where there hasbeen amplification of one or more segments of one or more targetsequences.

As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

As used herein, the term “antisense” is used in reference to DNA or RNAsequences that are complementary to a specific DNA or RNA sequence(e.g., mRNA). Included within this definition are antisense RNA(“asRNA”) molecules involved in gene regulation by bacteria. AntisenseRNA may be produced by any method, including synthesis by splicing thegene(s) of interest in a reverse orientation to a viral promoter whichpermits the synthesis of a coding strand. Once introduced into anembryo, this transcribed strand combines with natural mRNA produced bythe embryo to form duplexes. These duplexes then block either thefurther transcription of the mRNA or its translation. In this manner,mutant phenotypes may be generated. The term “antisense strand” is usedin reference to a nucleic acid strand that is complementary to the“sense” strand. The designation (−) (i.e., “negative”) is sometimes usedin reference to the antisense strand, with the designation (+) sometimesused in reference to the sense (i.e., “positive”) strand.

The terms “in operable combination,” “in operable order,” and “operablylinked” as used herein refer to the linkage of nucleic acid sequences insuch a manner that a nucleic acid molecule capable of directing thetranscription of a given gene and/or the synthesis of a desired proteinmolecule is produced. The term also refers to the linkage of amino acidsequences in such a manner so that a functional protein is produced.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecontaminant nucleic acid with which it is ordinarily associated in itsnatural source. Isolated nucleic acid is such present in a form orsetting that is different from that in which it is found in nature. Incontrast, non-isolated nucleic acids as nucleic acids such as DNA andRNA found in the state they exist in nature. For example, a given DNAsequence (e.g., a gene) is found on the host cell chromosome inproximity to neighboring genes; RNA sequences, such as a specific mRNAsequence encoding a specific protein, are found in the cell as a mixturewith numerous other mRNAs that encode a multitude of proteins. However,isolated nucleic acid encoding a given protein includes, by way ofexample, such nucleic acid in cells ordinarily expressing the givenprotein where the nucleic acid is in a chromosomal location differentfrom that of natural cells, or is otherwise flanked by a differentnucleic acid sequence than that found in nature. The isolated nucleicacid, oligonucleotide, or polynucleotide may be present insingle-stranded or double-stranded form. When an isolated nucleic acid,oligonucleotide or polynucleotide is to be utilized to express aprotein, the oligonucleotide or polynucleotide will contain at a minimumthe sense or coding strand (i.e., the oligonucleotide or polynucleotidemay be single-stranded), but may contain both the sense and anti-sensestrands (i.e., the oligonucleotide or polynucleotide may bedouble-stranded).

As used herein, the term “purified” or “to purify” refers to the removalof contaminants from a sample. For example, antibodies are purified byremoval of contaminating non-immunoglobulin proteins; they are alsopurified by the removal of immunoglobulin that does not bind to thetarget molecule. The removal of non-immunoglobulin proteins and/or theremoval of immunoglobulins that do not bind to the target moleculeresults in an increase in the percent of target-reactive immunoglobulinsin the sample. In another example, recombinant polypeptides areexpressed in bacterial host cells and the polypeptides are purified bythe removal of host cell proteins; the percent of recombinantpolypeptides is thereby increased in the sample.

The term “recombinant DNA molecule” as used herein refers to a DNAmolecule that is comprised of segments of DNA joined together by meansof molecular biological techniques. For example, “recombinant DNAvector” refers to DNA sequences containing a desired coding sequence andappropriate DNA sequences necessary for the expression of the operablylinked coding sequence in a particular host organism.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule that is expressed from a recombinantDNA molecule.

The term “native protein” as used herein to indicate that a protein doesnot contain amino acid residues encoded by vector sequences; that is thenative protein contains only those amino acids found in the protein asit occurs in nature. A native protein may be produced by recombinantmeans or may be isolated from a naturally occurring source.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid.

The term “transgene” as used herein refers to a foreign gene that isplaced into an organism by, for example, introducing the foreign geneinto newly fertilized eggs or early embryos. The term “foreign gene”refers to any nucleic acid (e.g., gene sequence) that is introduced intothe genome of an animal by experimental manipulations and may includegene sequences found in that animal so long as the introduced gene doesnot reside in the same location as does the naturally-occurring gene.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.” Vectorsare often derived from plasmids, bacteriophages, or plant or animalviruses.

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes usually include a promoter, anoperator (optional), and a ribosome binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

Embryonal cells at various developmental stages can be used to introducetransgenes for the production of transgenic animals. Different methodsare used depending on the stage of development of the embryonal cell.The zygote is the best target for micro-injection. In the mouse, themale pronucleus reaches the size of approximately 20 micrometers indiameter which allows reproducible injection of 1-2 picoliters (pl) ofDNA solution. The use of zygotes as a target for gene transfer has amajor advantage in that in most cases the injected DNA will beincorporated into the host genome before the first cleavage (Brinster etal., Proc. Natl. Acad. Sci. USA 82:4438-4442 [1985]). As a consequence,all cells of the transgenic non-human animal will carry the incorporatedtransgene. This will, in general, also be reflected in the efficienttransmission of the transgene to offspring of the founder since 50% ofthe germ cells will harbor the transgene. Micro-injection of zygotes isthe preferred method for incorporating transgenes in practicing theinvention. U.S. Pat. No. 4,873,191 describes a method for themicro-injection of zygotes; the disclosure of this patent isincorporated herein in its entirety.

Retroviral infection can also be used to introduce transgenes into ananimal. The developing embryo can be cultured in vitro to the blastocyststage. During this time, the blastomeres can be targets for retroviralinfection (Janenich, Proc. Natl. Acad. Sci. USA 73:1260-1264 [1976]).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Hogan et al., in Manipulatingthe Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. [1986]). The viral vector system used to introduce thetransgene is typically a replication-defective retrovirus carrying thetransgene (D. Jahner et al., Proc. Natl. Acad Sci. USA 82:6927-693[1985]). Transfection is easily and efficiently obtained by culturingthe blastomeres on a monolayer of virus-producing cells (Van der Putten,supra; Stewart, et al., EMBO J. 6:383-388 [1987]). Alternatively,infection can be performed at a later stage. Virus or virus-producingcells can be injected into the blastocoele (D. Jahner et al., Nature298:623-628 [1982]). Most of the founders will be mosaic for thetransgene since incorporation occurs only in a subset of cells that formthe transgenic animal. Further, the founder may contain variousretroviral insertions of the transgene at different positions in thegenome that generally will segregate in the offspring. In addition, itis also possible to introduce transgenes into the germline, albeit withlow efficiency, by intrauterine retroviral infection of the midgestationembryo (Jahner et al., supra [1982]). Additional means of usingretroviruses or retroviral vectors to create transgenic animals known tothe art involves the micro-injection of retroviral particles ormitomycin C-treated cells producing retrovirus into the perivitellinespace of fertilized eggs or early embryos (PCT International ApplicationWO 90/08832 [1990], and Haskell and Bowen, Mol. Reprod. Dev., 40:386[1995]).

A third type of target cell for transgene introduction is the embryonalstem (ES) cell. ES cells are obtained by culturing pre-implantationembryos in vitro under appropriate conditions (Evans et al., Nature292:154-156 [1981]; Bradley et al., Nature 309:255-258 [1984]; Gossleret al., Proc. Acad. Sci. USA 83:9065-9069 [1986]; and Robertson et al.,Nature 322:445-448 [1986]). Transgenes can be efficiently introducedinto the ES cells by DNA transfection by a variety of methods known tothe art including calcium phosphate co-precipitation, protoplast orspheroplast fusion, lipofection and DEAE-dextran-mediated transfection.Transgenes may also be introduced into ES cells by retrovirus-mediatedtransduction or by micro-injection. Such transfected ES cells canthereafter colonize an embryo following their introduction into theblastocoel of a blastocyst-stage embryo and contribute to the germ lineof the resulting chimeric animal (for review, See, Jaenisch, Science240:1468-1474 [1988]). Prior to the introduction of transfected ES cellsinto the blastocoel, the transfected ES cells may be subjected tovarious selection protocols to enrich for ES cells that have integratedthe transgene assuming that the transgene provides a means for suchselection. Alternatively, the polymerase chain reaction may be used toscreen for ES cells that have integrated the transgene. This techniqueobviates the need for growth of the transfected ES cells underappropriate selective conditions prior to transfer into the blastocoel.

The terms “overexpression” and “overexpressing” and grammaticalequivalents, are used in reference to levels of mRNA to indicate a levelof expression approximately 3-fold higher than that typically observedin a given tissue in a control or non-transgenic animal. Levels of mRNAare measured using any of a number of techniques known to those skilledin the art including, but not limited to Northern blot analysis.Appropriate controls are included on the Northern blot to control fordifferences in the amount of RNA loaded from each tissue analyzed (e.g.,the amount of 28S rRNA, an abundant RNA transcript present atessentially the same amount in all tissues, present in each sample canbe used as a means of normalizing or standardizing the mRNA-specificsignal observed on Northern blots). The amount of mRNA present in theband corresponding in size to the correctly spliced transgene RNA isquantified; other minor species of RNA which hybridize to the transgeneprobe are not considered in the quantification of the expression of thetransgenic mRNA.

The term “transfection” as used herein refers to the introduction offoreign DNA into eukaryotic cells. Transfection may be accomplished by avariety of means known to the art including calcium phosphate-DNAco-precipitation, DEAE-dextran-medicated transfection,polybrene-mediated transfection, electroporation, microinjection,liposome fusion, lipofection, protoplast fusion, retroviral infection,and biolistics.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell whichhas stably integrated foreign DNA into the genomic DNA.

The term “transient transfection” or “transiently transfected” refers tothe introduction of foreign DNA into a cell where the foreign DNA failsto integrate into the genome of the transfected cell. The foreign DNApersists in the nucleus of the transfected cell for several days. Duringthis time the foreign DNA is subject to the regulatory controls thatgovern the expression of endogenous genes in the chromosomes. The term“transient transfectant” refers to cells which have taken up foreign DNAbut have failed to integrate this DNA.

The term “calcium phosphate co-precipitation” refers to a technique forthe introduction of nucleic acids into a cell. The uptake of nucleicacids by cells is enhanced when the nucleic acid is presented as acalcium phosphate-nucleic acid co-precipitate. The original technique ofGraham and van der Eb (Graham and van der Eb, Virol., 52:456 [1973]),has been modified by several groups to optimize conditions forparticular types of cells. The art is well aware of these numerousmodifications.

As used herein, the term “selectable marker” refers to the use of a genethat encodes an enzymatic activity that confers the ability to grow inmedium lacking what would otherwise be an essential nutrient (e.g. theHIS3 gene in yeast cells); in addition, a selectable marker may conferresistance to an antibiotic or drug upon the cell in which theselectable marker is expressed. Selectable markers may be “dominant”; adominant selectable marker encodes an enzymatic activity that can bedetected in any eukaryotic cell line. Examples of dominant selectablemarkers include the bacterial aminoglycoside 3′ phosphotransferase gene(also referred to as the neo gene) that confers resistance to the drugG418 in mammalian cells, the bacterial hygromycin G phosphotransferase(hyg) gene that confers resistance to the antibiotic hygromycin and thebacterial xanthine-guanine phosphoribosyl transferase gene (alsoreferred to as the gpt gene) that confers the ability to grow in thepresence of mycophenolic acid. Other selectable markers are not dominantin that there use must be in conjunction with a cell line that lacks therelevant enzyme activity. Examples of non-dominant selectable markersinclude the thymidine kinase (tk) gene that is used in conjunction withtk⁻ cell lines, the CAD gene which is used in conjunction withCAD-deficient cells and the mammalian hypoxanthine-guaninephosphoribosyl transferase (hprt) gene which is used in conjunction withhprt⁻ cell lines. A review of the use of selectable markers in mammaliancell lines is provided in Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, NewYork (1989) pp.16.9-16.15.

As used herein, the term “cell culture” refers to any in vitro cultureof cells. Included within this term are continuous cell lines (e.g.,with an immortal phenotype), primary cell cultures, finite cell lines(e.g., non-transformed cells), and any other cell population maintainedin vitro.

The term “test compound” refers to any chemical entity, pharmaceutical,drug, and the like that can be used to treat or prevent a disease,illness, sickness, or disorder of bodily function. Test compoundscomprise both known and potential therapeutic compounds. A “knowntherapeutic compound” refers to a therapeutic compound that has beenshown (e.g., through animal trials or prior experience withadministration to humans) to be effective in such treatment orprevention.

As used herein, the terms “host,” “expression host,” and “transformant”refer to organisms and/or cells which harbor an exogenous DNA sequence(e.g., via transfection), an expression vector or vehicle, as well asorganisms and/or cells that are suitable for use in expressing arecombinant gene or protein. It is not intended that the presentinvention be limited to any particular type of cell or organism. Indeed,it is contemplated that any suitable organism and/or cell will find usein the present invention as a host.

As used herein, the term “antigen” refers to any agent (e.g., anysubstance, compound, molecule [including macromolecules], or othermoiety), that is recognized by an antibody, while the term “immunogen”refers to any agent (e.g., any substance, compound, molecule [includingmacromolecules], or other moiety) that can elicit an immunologicalresponse in an individual. These terms may be used to refer to anindividual macromolecule or to a homogeneous or heterogeneous populationof antigenic macromolecules. It is intended that the term encompassesprotein molecules or at least one portion of a protein molecule, whichcontains one or more epitopes. In many cases, antigens are alsoimmunogens, thus the term “antigen” is often used interchangeably withthe term “immunogen.” The substance may then be used as an antigen in anassay to detect the presence of appropriate antibodies in the serum ofthe immunized animal.

The term “antigenic determinant” as used herein refers to that portionof an antigen that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the “immunogen” used to elicitthe immune response) for binding to an antibody.

The terms “specific binding” or specifically binding” when used inreference to the interaction of an antibody and a protein or peptidemeans that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A,” the presence of aprotein containing epitope A (or free, unlabelled A) in a reactioncontaining labelled “A” and the antibody will reduce the amount oflabelled A bound to the antibody.

The term “monovalent” when used in reference to a vaccine refers to avaccine which is capable of provoking an immune response in a hostanimal directed against a single type of antigen. In contrast, a“multivalent” vaccine provokes an immune response in a host animaldirected against several (i.e., more than one) toxins and/or enzymesassociated with disease (e.g., glycoprotease and/or neuraminidase). Itis not intended that the vaccine be limited to any particular organismor immunogen.

The present invention further contemplates immunization with or withoutadjuvant. As used herein, the term “adjuvant” is defined as a substanceknown to increase the immune response to other antigens whenadministered with other antigens. If adjuvant is used, it is notintended that the present invention be limited to any particular type ofadjuvant—or that the same adjuvant, once used, be used all the time. Itis contemplated that adjuvants may be used either separately or incombination. The present invention contemplates all types of adjuvant,including but not limited to agar beads, aluminum hydroxide or phosphate(alum), Incomplete Freund's Adjuvant, as well as Quil A adjuvantcommercially available from Accurate Chemical and ScientificCorporation, Gerbu adjuvant also commercially available (GmDP; C.C.Biotech Corp.), and bacterin (i.e., killed preparations of bacterialcells). It is further contemplated that the vaccine comprise at leastone “excipient” (i.e., a pharmaceutically acceptable carrier orsubstance) suitable for administration to a human or other animalsubject. It is intended that the term “excipient” encompass liquids, aswell as solids, and colloidal suspensions.

As used herein the term “immunogenically-effective amount” refers tothat amount of an immunogen required to invoke the production ofprotective levels of antibodies in a host upon vaccination.

The term “protective level,” when used in reference to the level ofantibodies induced upon immunization of the host with an immunogen meansa level of circulating antibodies sufficient to protect the host fromchallenge with a lethal dose of the organism or other antigenic material(e.g., toxins, etc.).

A “B cell epitope” generally refers to the site on an antigen to which aspecific antibody molecule binds. The identification of epitopes whichare able to elicit an antibody response is readily accomplished usingtechniques well known in the art (See e.g., Geysen et al. Proc. Natl.Acad. Sci. USA 81:3998-4002 [1984], for general method of rapidlysynthesizing peptides to determine the location of immunogenic epitopesin a given antigen; U.S. Pat. No. 4,708,871 for procedures foridentifying and chemically synthesizing epitopes of antigens; and Geysenet al., Mol. Immunol., 23:709-715 [1986] for a technique for identifyingpeptides with high affinity for a given antibody).

A “T cell epitope” refers generally to those features of a peptidestructure capable of inducing a T cell response. In this regard, it isaccepted in the art that T cell epitopes comprise linear peptidedeterminants that assume extended conformations within thepeptide-binding cleft of MHC molecules, (See, Unanue et al., Science236:551-557 [1987]). As used here, a T cell epitope is generally apeptide having about 3-5, preferably 5-10 or more, amino acid residues.

The term “self antigen” or “autoantigen,” means an antigen or a moleculecapable of being recognized during an immune response as self (i.e., anantigen that is normally part of the individual). This is in contrast toantigens which are foreign, or exogenous, and are thus not normally partof the individual's antigenic makeup.

As used herein, the term “autoimmune disease” means a set of sustainedorgan-specific or systemic clinical symptoms and signs associated withaltered immune homeostasis that is manifested by qualitative and/orquantitative defects of expressed autoimmune repertoires. Autoimmunediseases are characterized by antibody or cytotoxic immune responses toepitopes on self antigens found in the diseased individual. The immunesystem of the individual then activates an inflammatory cascade aimed atcells and tissues presenting those specific self antigens. Thedestruction of the antigen, tissue, cell type, or organ attacked by theindividual's own immune system gives rise to the symptoms of thedisease. Clinically significant autoimmune diseases include, forexample, rheumatoid arthritis, multiple sclerosis, juvenile-onsetdiabetes, systemic lupus erythematosus (SLE), autoimmune uveoretinitis,autoimmune vasculitis, bullous pemphigus, myasthenia gravis, autoimmunethyroiditis or Hashimoto's disease, Sjogren's syndrome, granulomatousorchitis, autoimmune oophoritis, Crohn's disease, sarcoidosis, rheumaticcarditis, ankylosing spondylitis, Grave's disease, and autoimmunethrombocytopenic purpura.

Another aspect of cellular immunity involves an antigen-specificresponse by helper T lymphocytes (T_(H) cells). T_(H) cells act to helpstimulate the function, and focus the activity of, nonspecific effectorcells against cells displaying peptide antigens in association with MHCmolecules on their surface. In addition, various subsets of T_(H) cellsproduce distinct cytokines in response to antigenic stimulation.Particularly, antigenic stimulation of naive T_(H) cells leads todifferentiation of the lymphocyte cells into subsets termed “T_(H)1” and“T_(H)2” which have relatively restricted cytokine production profilesand effector functions. T_(H)1 cells secrete IL-2 and IFN-γ, and are theprincipal effectors of cell-mediated immunity against intracellularmicrobes and of delayed type hypersensitivity (DTH) reactions. Antibodyisotypes stimulated by T_(H)1 cells are effective at activatingcomplement and opsonizing antigens for phagocytosis. T_(H)2 cellsproduce IL-4 (which stimulates IgE antibody production), IL-5(eosinophil-activating factor), and IL-10 and IL-13 (which suppresscell-mediated immunity). Thus, the nature of an immune response can becharacterized by the profile of antigen-specific lymphocytes that arestimulated by the immunogen, and can be referred to as a “T_(H)1-like”or a “T_(H)2-like” immune response.

The ability of a particular antigen to stimulate a cell-mediatedimmunological response may be determined by a number of assays, such asby lymphoproliferation (lymphocyte activation) assays, CTL cytotoxiccell assays such as chromium-release assays, or by assaying for Tlymphocytes specific for the antigen in a sensitized subject. Suchassays are well known in the art (See e.g., Erickson et al., J.Immunol., 151:4189-4199 [1993], and Doe et al., Eur. J. Immunol.,24:2369-2376 [1994]).

The term “sample” in the present specification and claims is used in itsbroadest sense. On the one hand it is meant to include a specimen orculture (e.g., microbiological cultures). On the other hand, it is meantto include both biological and environmental samples.

Biological samples may be animal, including human, fluid, solid (e.g.,stool) or tissue, as well as liquid and solid food and feed products andingredients such as dairy items, vegetables, meat and meat by-products,and waste. Biological samples may be obtained from all of the variousfamilies of domestic animals, as well as feral or wild animals,including, but not limited to, such animals as ungulates, bear, fish,lagamorphs, rodents, etc.

Environmental samples include environmental material such as surfacematter, soil, water, and industrial samples, as well as samples obtainedfrom food and dairy processing instruments, apparatus, equipment,utensils, disposable, and non-disposable items. These examples are notto be construed as limiting the sample types applicable to the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention relates to Zic related genes.In particular, three Zic related genes, Zic1, Zic2, and Zic3 areprovided, identified, and compared with respect to their expressionpatterns and effects of overexpression. The expression patterns ofXenopus Zic1, Zic2, and Zic3 were found to be distinct, although allthree genes expressed in the prospective neural plate region ingastrula. Zic1 and Zic2 were functionally related to Zic3 in that thesegenes induce neural and neural crest tissues when overexpressed.

Xenopus Zic1 and Zic2 were also found to be very similar to mouse Zic1and Zic2 in the protein coding region including the zinc finger domain.In early gastrula, Zic1 expression was restricted to the prospectiveneural plate region whereas Zic2 was expressed widely in the ectoderm.Enhanced neural and neural crest-derived tissue formation were observedin Zic1 or Zic2 overexpressed embryos, as well as in the neural andneural crest marker induction in Zic1 or Zic2 overexpressed animal capexplants. These findings suggest that Zic1 and Zic2 have essentially thesame properties as Zic3, and that the Xenopus Zic family actcooperatively in the initial phase of neural and neural crestdevelopment.

The Detailed Description of the Invention is divided into seven parts:I) Cloning of Neurogenesis Genes; II) Preparation of Recombinant Vectorsand Transformants; III) Analysis of Gene Expression; IV) ProteinProduction; V) Antibodies Against Zic Proteins; VI) Therapeutic Agentsand Agents for Gene Therapy for Nervous Diseases; and VII) Isolation andCharacterization of Zic1 and Zic2.

I. Cloning of Neurogenesis Genes

mRNA can be prepared by conventional methods. For example, tissue orcells are treated with a guanidine reagent, phenol reagent or the liketo obtain the total RNA. Subsequently, poly(A+)RNA (mRNA) is obtainedtherefrom by the affinity column method using oligo dT-cellulose or polyU-Sepharose carried on Sepharose 2B or by the batch method. Further, theresultant poly(A+)RNA may be further fractionated by sucrose gradientcentrifugation or the like.

A single-stranded cDNA is synthesized using the thus obtained mRNA as atemplate, an oligo(dT) primer and a reverse transcriptase. Then, adouble-stranded cDNA is synthesized from the resultant single-strandedcDNA. The resultant double-stranded cDNA is integrated into anappropriate cloning vector to prepare a recombinant vector. A cDNAlibrary can be obtained by transforming Escherichia coli or the likewith the resultant recombinant vector and selecting the transformantusing tetracycline or ampicillin resistance as an indicator.

The transformation of E. coli can be performed by the method of Hanahan(Hanahan, J. Mol. Biol. 166: 557-580 [1983]) or the like. Briefly, amethod in which a recombinant vector is added to competent cellsprepared under the co-existence of calcium chloride, magnesium chlorideor rubidium chloride may be used. When a plasmid is used as a vector,the plasmid should contain a drug resistance gene such as tetracyclineor ampicillin resistance. Alternatively, a cloning vector other thanplasmids (e.g. a phage or the like) may be used.

As a screening method to select clones containing the DNA of interestfrom the resultant transformants, a method may be given, for example, inwhich a sense primer and an anti-sense primer corresponding to the aminoacid sequence of the zinc finger motif of the mouse Zic gene family aresynthesized and a polymerase chain reaction (PCR) is performed usingthese primers.

As a template DNA to be used in the above PCR, a cDNA which issynthesized from the above-described mRNA by reverse transcription maybe given. As primers, 5′-GAGAACCTCAAGATCCACAA-3′ (SEQ ID NO: 5)synthesized based on Glu Asn Leu Lys Ile His Lys (SEQ ID NO: 3) may beused for the same strand; and 5′-TT(C/T)CCATG(A/G)ACCTTCATGTG-3′ (SEQ IDNO: 6) synthesized based on His Met Lys Val His Glu Glu (SEQ ID NO: 4)may be used for the anti-sense strand, for example. However, the presentinvention is not limited to the use of these primers.

The amplified DNA fragment obtained by the above procedures is labelledwith ³²P, ³⁵S, biotin or the like, to obtain a probe. This probe ishybridized to a nitrocellulose filter on which the DNA of thetransformant is denatured and fixed. Then, screening can be performed bysearching for positive clones.

For the resultant clone, the nucleotide sequence is determined. Thissequencing is performed by conventional methods such as the chemicalmodification method of Maxam-Gilbert or the dideoxynucleotide chaintermination method using M13 phage. In preferred embodiments, thesequencing is carried out with an automated DNA sequencer (e.g.,PerkinElmer Model 373A DNA Sequencer).

SEQ ID NO: 1 illustrates by example of a nucleotide sequence for theZic3 gene of the present invention, and SEQ ID NO: 2 illustrates byexample an amino acid sequence for the associated protein. The presentinvention contemplates variation of this amino acid sequence. Proteinsfind use with the present invention as long as the protein hasneurogenesis inducing activity, and in particular early neurogenesisinducing activity. Thus, the amino acid sequence may have one or moremutations, such as deletions, substitutions, or additions, of at leastone amino acid.

For example, at least 1 amino acid, preferably 1 to about 10 aminoacids, more preferably 1 to 5 amino acids may be deleted in the aminoacid sequence shown in SEQ ID NO:2; or at least 1 amino acid, preferably1 to about 10 amino acids, more preferably 1 to 5 amino acids may beadded to the amino acid sequence shown in SEQ ID NO:2; or at least 1amino acid, preferably 1 to about 10 amino acids, more preferably 1 to 5amino acids may be substituted with other amino acid(s) in the aminoacid sequence shown in SEQ ID NO:2.

Accordingly, a gene coding for a polypeptide having the amino acidsequence into which the above-mentioned mutation has been introduced isincluded in the gene of the invention as long as it has neurogenesisinducing activity (e.g., early neurogenesis inducing activity). Also, aDNA which can hybridize with the gene described above under stringentconditions is provided by the present invention. For example, in someembodiments of the present invention, stringent conditions refers tothose conditions in which sodium concentration is 600-900 mM andtemperature is 60-68° C., preferably 65° C.

The introduction of a mutation into a gene may be performed byconventional methods such as the method of Kunkel, the gaped duplexmethod, or variations thereof using a mutation introducing kit (e.g.,Mutant-K [Takara] or Mutant-G [Takara]) utilizing site-specificmutagenesis or using a LA PCR in vitro Mutagenesis Series Kitmanufactured by Takara, or the like.

Once the nucleotide sequence of the gene of the invention has beendetermined definitely, the gene of the invention may be obtained bychemical synthesis, by PCR using the cDNA or genomic DNA of the gene ofthe invention as a template, or by hybridization of a DNA fragmenthaving the above nucleotide sequence as a probe.

II. Preparation of a Recombinant Vector and a Transformant

A. Preparation of a Recombinant Vector

The recombinant vector of the invention may be obtained by ligating(i.e., inserting) the gene of the invention to an appropriate vector.The vector into which the gene of the invention is to be inserted is notparticularly limited as long as it is replicable in a desired host. Forexample, plasmid DNA, phage DNA or the like may be used.

Specific examples of plasmid DNA include E. coli-derived plasmids (e.g.,pBR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis-derivedplasmids (e.g., pUB110, pTP5, etc.) and yeast-derived plasmids (e.g.,YEp13, YEp24, YCp50, etc.). Specific examples of phage DNA include Aphage and the like. Further, an animal virus vector such as retrovirusor vaccinia virus; or an insect virus vector such as baculovirus mayalso be used.

For insertion of the gene of the invention into a vector, a method maybe employed in which the purified DNA is digested with an appropriaterestriction enzyme and then inserted into the restriction site or themulti-cloning site of an appropriate vector DNA for ligation to thevector.

The gene of the invention should be operably linked to the vector. Forthis purpose, the vector of the invention may contain, if desired, ciselements such as an enhancer, splicing signal, poly(A) addition signal,selection marker, ribosome binding sequence (SD sequence) or the like inaddition to a promoter and the gene of the invention. As the selectionmarker, dihydrofolate reductase gene, ampicillin resistance gene,neomycin resistance gene, or the like may be used.

B. Preparation of a Transformant

In some embodiments of the present invention, the transformant isobtained by introducing the recombinant vector of the invention into ahost so that the gene of interest is expressed. The host is notparticularly limited as long as it can express the DNA of the invention.Specific examples of the host include Escherichia bacteria such as E.coli; Bacillus bacteria such as Bacillus subtilis; Pseudomonas bacteriasuch as Pseudomonas putida; Rhizobium bacteria such as Rhizobiummeliloti; yeasts such as Saccharomyces cerevisiae, Schizosaccharomycespombe; animal cells such as COS cells, CHO cells; or insect cells suchas Sf9 and Sf21 cells.

When a bacterium such as E. coli is used as the host, the recombinantvector of the invention is capable of autonomous replication in the hostand, at the same time, it is constituted preferably by a promoter, aribosome binding sequence, the gene of the invention and a transcriptiontermination sequence. The vector may also contain a gene to control thepromoter.

Examples of E. coli that find use with the present invention include,but are not limited to, K12 or DH1 strains. Examples of Bacillussubtilis that find use with the present invention include, but are notlimited to, MI 114 or 207-21 strains.

As the promoter, any promoter may be used as long as it can direct theexpression of the gene of interest in a host such as E. coli. Forexample, an E. coli- or phage-derived promoter such as trp promoter, lacpromoter, P_(L) promoter or P_(R) promoter may be used. An artificiallydesigned and altered promoter such as tac promoter may also be used.

As a method for introducing the recombinant vector into a bacterium, anymethod of DNA introduction into bacteria may be used. For example, amethod using calcium ions (Cohen et al., Proc. Natl. Acad. Sci., USA,69: 2110-2114 [1972]) electroporation, or the like may be used.

In some embodiments of the present invention, when a yeast is used asthe host, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichiapastoris or the like is used. In this case, the promoter to be used isnot particularly limited. Any promoter may be used as long as it candirect the expression of the gene of interest in yeast. For example,gal1 promoter, gal10 promoter, heat shock protein promoter, MF α1promoter, PH05 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1promoter or the like may be used.

As a method for introducing the recombinant vector into the yeast, anymethod of DNA introduction into yeasts may be used. For example,electroporation (Becker et al., Methods Enzymol., 194: 182-187 [1990]),the spheroplast method (Hinnen et al., Proc. Natl. Acad. Sci., USA, 75:1929-1933 [1978]), the lithium acetate method (Itoh, Bacteriol., 153:163168 [19893]) or the like may be used.

When an animal cell is used as the host, simian COS-7 or Vero cells,Chinese hamster ovary cells (CHO cells), mouse L cells, rat GH3 cells,human FL cells or the like may be used, although a variety of othercells find use with the present invention. As a promoter, SRα promoter,SV40 promoter, LTR promoter, CMV promoter or the like may be used. Theearly gene promoter of human cytomegalovirus may also be used.

As a method for introducing the recombinant vector into the animal cell,electroporation, the calcium phosphate method, lipofection or the likemay be used.

When an insect cell is used as the host, Sf9 cells, Sf21 cells or thelike may be used. As a method for introducing the recombinant vectorinto the insect cell, the calcium phosphate method, lipofection,electroporation or the like may be used.

The recombinant vector of the invention incorporated in E. coli DH5(designation: Escherichia coli pXenopus Zic3) was deposited at theNational Institute of Bioscience and Human Technology, Agency ofIndustrial Science and Technology (1-3, Higashi 1-Chome, Tsukuba City,Ibaraki Pref., Japan) as FERM BP-6519 under the Budapest Treaty on Mar.26, 1998.

III. Analysis of Gene Expression

Since the gene of the invention has neurogenesis inducing activity, theexpression of this gene can be examined by using embryos of specificdevelopmental stages.

The time of expression of the Zic3 gene of the invention in the embryocan be confirmed by analyzing, for example, expression of the mRNA orthe protein in embryos of individual developmental stages. For example,as a method for confirming expression of Zic3 mRNA, RT-PCR, or northernanalysis may be used; as a method for confirming expression of ZIC3protein, western analysis using an antibody against this protein may beused.

Further, the distribution of Zic3 expression in the embryo can beconfirmed by analyzing the mRNA by in situ hybridization or the like, orby analyzing the protein by immunohistochemical techniques using anantibody. In situ hybridization can be performed, for example, asdescribed previously (Chitnis et al., Nature 375: 761-766 [1995]) bystaining the embryo with digoxigenin or a radioisotope labelled RNAprobe.

IV. Protein Production

In some embodiments of the present invention, for example, the proteinof the invention is a protein having the amino acid sequence encoded bythe Zic3 gene of the invention, or a protein which has the above aminoacid sequence having the mutation of at least at 1 amino acid and whichhas neurogenesis inducing activity. This protein is also called “ZIC3protein”.

ZIC3 protein of the present invention can be obtained by culturing thetransformant described above and recovering the protein from theresultant culture. The term “culture” includes any of the followingmaterials: culture supernatant, cultured cells, cultured microorganisms,or crushed cells or microorganisms. The cultivation of the transformantof the invention in a medium is carried out by conventional methods usedfor culturing a host.

As a medium to culture the transformant obtained from a microorganismhost such as E. coli or yeast, either a natural or a synthetic mediummay be used as long as it contains carbon sources, nitrogen sources andinorganic salts assimilable by the microorganism and is capable ofeffective cultivation of the transformant. As carbon sources,carbohydrates such as glucose, fructose, sucrose, starch; organic acidssuch as acetic acid, propionic acid; and alcohols such as ethanol andpropanol may be used. As nitrogen sources, ammonia; ammonium salts ofinorganic or organic acids such as ammonium chloride, ammonium sulfate,ammonium acetate, ammonium phosphate; other nitrogen-containingcompounds; Peptone; meat extract; corn steep liquor and the like may beused. As inorganic substances, potassium dihydrogen phosphate,dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate,sodium chloride, iron(II) sulfate, manganese sulfate, copper sulfate,calcium carbonate and the like may be used.

In preferred embodiments, the cultivation is carried out under aerobicconditions (such as shaking culture or aeration agitation culture) at37° C. for 6 to 24 hrs. During the cultivation, the pH is maintained at7.0 to 7.5. The pH adjustment is carried out using an inorganic ororganic salt, an alkali solution or the like. During the cultivation, anantibiotic such as ampicillin or tetracycline may be added to the mediumif necessary.

When a microorganism transformed with an expression vector using aninducible promoter is cultured, an inducer may be added to the medium ifnecessary. For example, when a microorganism transformed with anexpression vector using Lac promoter is cultured,isopropyl-O-D-thiogalactopyranoside (IPTG) or the like may be added.When a microorganism transformed with an expression vector using trppromoter is cultured, indoleacetic acid (IAA) or the like may be added.

As a medium to culture a transformant obtained from an animal cell as ahost, commonly used RPMI 1640 medium or DMEM medium, or one of thesemedia supplemented with fetal bovine serum, etc. may be used. Usually,the cultivation is carried out in the presence 5% CO₂ at 37° C. for 1 to30 days. During the cultivation, an antibiotic such as kanamycin orpenicillin may be added to the medium if necessary.

After the cultivation, ZIC3 protein of the invention is extracted bydisrupting the microorganisms or cells if the protein is produced in themicroorganisms or cells. If ZIC3 protein of the invention is producedoutside of the microorganisms or cells, the culture fluid is useddirectly or is subjected to centrifugation to remove the microorganismsor cells. Thereafter, the resultant supernatant is subjected toconventional biochemical techniques used for isolating/purifying aprotein. These techniques include ammonium sulfate precipitation, gelchromatography, ion exchange chromatography, and affinitychromatography. These techniques may be used independently or in anappropriate combination to thereby isolate and purify ZIC3 protein ofthe invention from the above culture.

V. Antibody Against Zic Proteins

In the present invention, antibody against ZIC proteins of the inventioncan also be prepared. The term “antibody” means an antibody molecule asa whole which can bind to the peptide of the invention that is anantigen, or a fragment thereof (e.g., Fab or F(ab′)₂ fragment). Theantibody may be either polyclonal or monoclonal.

The antibody of the invention may be prepared by various methods. Suchmethods of antibody preparation are well known in the art (See e.g.,Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor LaboratoryPress [1989]).

A. Preparation of a Polyclonal Antibody Against the Protein of theInvention

The following description is provided for the ZIC3 protein and isapplicable to other ZIC proteins. In some embodiments of the presentinvention, ZIC3 protein of the invention is genetically engineered asdescribed above or a fragment thereof is administered as an antigen to amammal such as rat, mouse or rabbit. The dosage of the antigenadministered per animal is 0.1 to 10 mg when no adjuvant is used, and 1to 100 μg when an adjuvant is used. As an adjuvant, Freund's completeadjuvant (FCA), Freund's incomplete adjuvant (FIA), aluminium hydroxideadjuvant or the like may be used. Immunization is performed mainly byintravenous, subcutaneous or intraperitoneal injection. The interval ofimmunization is not particularly limited. In preferred embodiments,immunization is carried out one to 10 times, preferably 2 to 5 times, atintervals of several days to several weeks, preferably at intervals of 2to 5 weeks. Subsequently, 6 to 60 days after the final immunization,antibody titer is determined by, preferably, enzyme immunoassay (EIA),radioimmunoassay (RIA) or the like. Blood is collected from the animal,on the day when the maximum antibody titer is shown, to thereby obtainantiserum. When purification of an antibody from the antiserum isnecessary, the antibody is purified by appropriately selecting aconventional method such as ammonium sulfate salting out, ion exchangechromatography, gel filtration, affinity chromatography, or using thesemethods in combination.

B. Monoclonal Antibodies

(i) Recovery of Antibody-Producing Cells

In some embodiments of the present invention, ZIC3 protein of theinvention genetically engineered as described above or a fragmentthereof is administered as an antigen to a mammal such as rat, mouse orrabbit. The dosage of the antigen administered per animal is, forexample, 0.1 to 10 mg when no adjuvant is used, and 1 to 100 μg when anadjuvant is used. As an adjuvant, Freund's complete adjuvant (FCA),Freund's incomplete adjuvant (FIA), aluminium hydroxide adjuvant or thelike may be used. Immunization is performed mainly by intravenous,subcutaneous or intraperitoneal injection. The interval of immunizationis not particularly limited. In preferred embodiments, immunization iscarried out one to 10 times, preferably 2 to 5 times, at intervals ofseveral days to several weeks, preferably at intervals of 2 to 5 weeks.Subsequently, 1 to 10 days, preferably 3 days after the finalimmunization, antibody-producing cells are collected. Asantibody-producing cells, spleen cells, lymph node cells, peripheralblood cells, etc. may be enumerated. Among them, spleen cells and locallymph node cells are preferable.

(ii) Cell Fusion

In order to obtain hybridomas, cell fusion between antibody-producingcells and myeloma cells is performed. As the myeloma cells to be fusedto the antibody-producing cells, a commonly available cell strain of ananimal such as mouse may be used. Preferably, a cell strain to be usedfor this purpose is one which has drug selectivity, cannot survive inHAT selective medium (i.e., containing hypoxanthine, aminopterin andthymidine) when unfused, and can survive there only when fused toantibody-producing cells. As myeloma cells, mouse myeloma cell strainsincluding, but not limited to, P3X63Ag.8.U1(P3U1), Sp2/0, NS-1 may beused.

Subsequently, the myeloma cells and the antibody-producing cellsdescribed above are subjected to cell fusion. Briefly, 1×10⁹ cells/ml ofthe antibody-producing cells and 1×10⁸ cells/ml of the myeloma cells aremixed together in equal volumes in an animal cell culture medium such asserum-free DMEM or RPMI-1640, and reacted in the presence of a cellfusion promoting agent. In some embodiments, as the cell fusionpromoting agent, polyethylene glycol with an average molecular weight of1,500 Da may be used. Alternatively, the antibody-producing cells andthe myeloma cells may be fused in a commercial cell fusion apparatusutilizing electric stimulation (e.g., electroporation).

(iii) Selection and Cloning of a Hybridoma

A hybridoma of interest is selected from the cells after the cellfusion. As a method for this selection, the resultant cell suspension isappropriately diluted with fetal bovine serum-containing RPMI-1640medium or the like and then plated on microtiter plates at a density ofabout 2×10⁵ cells/well. A selective medium is added to each well. Then,the cells are cultured while appropriately exchanging the selectivemedium. As a result, about 14 days after the start of cultivation in theselective medium, the growing cells are obtained as hybridomas.

Subsequently, screening is performed as to whether the antibody ofinterest is present in the culture supernatant of the grown hybridomas.The screening of hybridomas may be performed by any of conventionalmethods. For example, a part of the culture supernatant of a well inwhich a hybridoma is grown is collected and subjected to enzymeimmunoassay or radioimmunoassay.

Cloning of the fused cell is performed by the limiting dilution methodor the like. Finally, the hybridoma of interest which is a monoclonalantibody-producing cell is established.

(iv) Recovery of the Monoclonal Antibody

In some embodiments of the present invention, a method for recoveringthe monoclonal antibody from the thus established hybridoma such asconventional cell culture methods or the abdominal dropsy formationmethod may be employed.

In the cell culture method, the hybridoma is cultured in an animal cellculture medium such as 10% fetal bovine serum-containing RPMI-1640medium, MEM medium or a serum-free medium under conventional cultureconditions (e.g., at 37° C. under 5% CO₂) for 2 to 10 days. Then, themonoclonal antibody is recovered from the culture supernatant.

In the abdominal dropsy formation method, about 1×10⁷ cells of thehybridoma is administered into the abdominal cavity of an animalsyngeneic to the mammal from which the myeloma cells were derived, tothereby propagate the hybridoma greatly. One to two weeks thereafter,the abdominal dropsy or serum is collected.

In the above-mentioned method of recovery of the antibody, ifpurification of the antibody is necessary, the antibody can be purifiedby appropriately selecting a conventional method such as ammoniumsulfate salting out, ion exchange chromatography, gel filtration,affinity chromatography, or using these methods in combination.

Once the polyclonal or monoclonal antibody is thus obtained, in someembodiments of the present invention, the antibody is bound to a solidcarrier as a ligand to thereby prepare an affinity chromatographycolumn. With this column, the peptides of the present invention can bepurified from the above mentioned source or other sources. Theseantibodies further find use in Western blotting to detect the proteinsof the present invention.

VI. Therapeutic Agents and Agents for Gene Therapy for Nervous Diseases

Since the proteins and the genes of the present invention haveneurogenesis inducing activity, they are useful as a therapeutic agentsand as agents for gene therapy, respectively, for nervous diseases. Insome embodiments of the present invention, the therapeutic agents or theagents for gene therapy of the present invention is administered to asubject orally or parenterally and systemically or locally.

When the protein or the gene of the present invention is used as atherapeutic agent or an agent for gene therapy for nervous diseases, thedisease to be treated is not particularly limited. For example, theproteins or the genes may be used, alone or in combination, for diseasesincluding, but not limited to, Alzheimer's disease, amyotrophic lateralsclerosis, spinocerebellar degeneration, Parkinson's disease, cerebralischemia or the like for the specific purpose of treatment orprevention. These diseases may be in the form of a single disease or maybe complicated by one of these diseases or by some disease other thanthose mentioned above. Any of such forms may be treated with theproteins or the genes of the invention.

In preferred embodiments of the present invention, when the therapeuticagent of the invention is administered orally, the agent may beformulated into a tablet, capsule, granule, powder, pill, troche,internal liquid agent, suspension, emulsion, syrup or the like.Alternatively, the therapeutic agent may be prepared into a dry productwhich is re-dissolved just before use. In preferred embodiments, whenthe therapeutic agent of the invention is administered parenterally, theagent may be formulated into a intravenous injection (including drops),intramuscular injection, intraperitoneal injection, subcutaneousinjection, suppository, or the like. Injections are supplied in the formof unit dosage ampules or multidosage containers. These formulations maybe prepared by conventional methods using appropriate excipients,fillers, binders, wetting agents, disintegrating agents, lubricatingagents, surfactants, dispersants, buffers, preservatives, dissolutionaids, antiseptics, flavoring/perfuming agents, analgesics, stabilizers,isotonicity inducing agents, etc. conventionally used in pharmaceuticalpreparations.

Each of the above-described formulations may contain pharmaceuticallyacceptable carriers or additives. Specific examples of such carriers oradditives include water, pharmaceutically acceptable organic solvents,collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinylpolymers, sodium alginate, water-soluble dextran, sodium carboxymethylamylose, pectin, xanthan gum, gum arabic, casein, gelatin, agar,glycerol, propylene glycol, polyethylene glycol, vaseline, paraffin,stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitoland lactose. One or a plurality of these additives are selected orcombined appropriately depending of the form of the preparation.

The dosage levels of the therapeutic agent of the invention will varydepending on the age of the subject, the route of administration and thenumber of times of administration and may be varied in a wide range.When an effective amount of the protein of the invention is administeredin combination with an appropriate diluent and a pharmaceuticallyacceptable carrier, the effective amount of the protein can be in therange from 0.01 to 1000 mg/kg per administration, although other amountsare contemplated, where appropriate. One skilled in the art is capableof determining the therapeutically effective amount appropriate anygiven circumstances. In some embodiments, the therapeutic agent isadministered once a day or in several dosages per day for at least oneday.

In some embodiments of the present invention, when the gene of theinvention is used as an agent for gene therapy for nervous diseases, thegene of the invention may be directly administered by injection.Alternatively, a vector incorporating the gene of the invention may beadministered. Specific examples of a suitable vector for this purposeinclude an adenovirus vector, adeno-associated virus vector, herpesvirus vector, vaccinia virus vector and retrovirus vector. The gene ofthe invention can be administered efficiently by using such a virusvector. Alternatively, the gene of the invention may be enclosed inphospholipid vesicles such as liposomes, and the resultant liposomes maybe administered to the subject. Briefly, since liposomes arebiodegradable material-containing closed vesicles, the gene of theinvention is retained in the internal aqueous layer and the lipidbilayer of liposomes by mixing the gene with the liposomes (i.e., aliposome-gene complex). Subsequently, when this complex is cultured withcells, the gene in the complex is taken into the cells (i.e.,lipofection). Then, the resultant cells may be administered by themethods described below.

In some embodiments of the present invention, as a method foradministering the agent for gene therapy of the invention, localadministration to tissues of the central nervous system (brain, spiralcord) may be performed in addition to conventional systemicadministration such as intravenous or intra-arterial administration.Further, an administration method combined with catheter techniques andsurgical operations may also be employed.

The dosage levels of the agent for gene therapy of the invention varydepending on the age, sex and conditions of the subject, the route ofadministration, the number of times of administration, and the type ofthe formulation, among other considerations. One skilled in the art iscapable of determining the therapeutically effective amount appropriateany given circumstances. Usually, it is appropriate to administer thegene of the invention in an amount of 0.1-100 mg/adult body/day,although other concentrations are contemplated, where appropriate.

According to the present invention, there are provided neurogenesisinducing proteins; a neurogenesis inducing genes (e.g., Zic1, Zic2,and/or Zic3) coding for the proteins; recombinant vectors comprising thegenes; transformants comprising the vectors; antibodies against theabove proteins; and therapeutic agents for nervous diseases. The Zicgenes of the invention find use as a diagnostic agents for nervousdiseases, as therapeutic agents for Alzheimer's disease and the like,and as probes to detect nervous diseases, among other applications.

VII. Isolation and Characterization of Zic1 and Zic2

A. Isolation of Xenopus Zic 1 and Zic2

To isolate additional Zic related genes in Xenopus, the Xenopus neurulacDNA library was further screened with the cDNA fragments generated byPCR. Two novel Xenopus Zic-related genes were identified (FIG. 8A, B). Acomparison of their predicted amino acid sequences to those of XenopusZic3, mouse Zic1, Zic2, Zic3, Zic4 and Drosophila Opa (Nakata et al.,supra; Aruga et al., J. Neurochem. 63: 1880-1890 [1994]; Aruga et al.,J. Biol. Chem. 271: 1043-1047 [1996]; Aruga et al., Gene 172: 291-294[1996], supra; Benedyk et al., supra) revealed that one was the mostsimilar to mouse Zic1, and the other was similar to Zic2. Thereforethese genes were designated Xenopus Zic1 (SEQ ID NO: 41) and Zic2 (SEQID NO: 43). Although significant homology was found in the entireprotein coding region, the most extensive homology was found in the zincfinger domains (98% between Xenopus Zic1 [SEQ ID NO: 42] and mouse Zic1,97% between Xenopus Zic2 [SEQ ID NO: 44] and mouse Zic2) (FIG. 1C). Inaddition, these novel genes showed significant similarity to Drosophilapair-rule gene, odd-paired (opa).

The zinc finger region, particularly from the 3rd to the 5th zinc fingermotif is highly similar to those of the Gli-Ci zinc finger proteins(Ruiz i Altaba, “Catching a Gli-mpse of Hedgehog,” Cell 90: 193-196[1997]), which mediate the hedgehog signal. A crystallographic analysisof Gli protein indicated that the same region actually interacts withthe DNA (Pavletich and Pabo, Pavletich, “Crystal structure of afive-finger GLI-DNA complex: new perspectives on zinc fingers,” Science261, 1701-1707 [1993]). Taken together with previous data indicatingthat mouse Zic1 can bind to the Gli-binding sequence (Aruga et al., J.Neurochem. 63: 1880-1890 [1994]), the current evidence suggests that thetwo protein families, Gli-Ci and Zic-Opa, can bind to highly similartarget sequences, although an understanding of the mechanism is notnecessary to practice the present invention and the present invention isnot limited to any particular mechanism.

In addition to the zinc finger domain, it is noted that there were shortdomains which are conserved in the Xenopus Zic, mouse Zic and Drosophilaopa genes. In particular, an amino acid sequence motif, FNSTRDFRXR (SEQID NO: 49), which was found in the N-terminal region, was highlyconserved (FIG. 8D).

B. Temporal Expression Profiles of Zic1 and Zic2 During XenopusDevelopment

RT-PCR analyses was performed to compare the temporal expressionpatterns of the Xenopus Zic genes (FIG. 9). Zic2 was maternallyexpressed, in contrast to Zic3, which was detected from late blastulabut not at earlier stages (Nakata et. al., 1997). Zic1 mRNA was detectedfrom the blastula stage (stage 8) and was peaked in the gastrula. Theexpression profile is similar to that of Zic3. Zic2 was continuouslyexpressed from the egg to the tailbud stage (stage 30) with an increasein expression at the early gastrula stage (stage 10).

C. Spatial Expression Patterns of Zic1 and Zic2 During XenopusEmbryogenesis

To determine the spatial expression patterns of Xenopus Zic1 and Zic2,whole mount in situ hybridization was performed. At the blastula stage(stage 9), Zic1 and Zic2 were expressed throughout the ectoderm (FIG.10A, M). At the gastrula stage (stage 10.5), Zic1 expression becamerestricted to the prospective neural plate, as observed for the Zic3expression (FIG. 10B, C; arrowhead, Nakata et. al., supra), whereas Zic2was expressed throughout the ectoderm in the gastrula (FIG. 10N, O).

During the late gastrula to neurula stages, both Zic1 and Zic2expression gradually diminished in the midline region of the neuralplate and increased in the anterior neural folds (FIG. 10D-F, P-R).However, Zic2 continued to be expressed in the posterior medial part ofthe neural plate (FIG. 10Q, arrowhead). At the neurula stage, stainingwas seen as four longitudinal lines in the trunk, similar to that ofZic3 (FIG. 10F, R, white and black arrowheads, Nakata, et. al., supra).

In the early tailbud stages (stages 22-23; FIG. 10G, H, S, T), Zic1 andZic2 were expressed in the dorsal forebrain, midbrain, and hindbrain(FIG. 10H, T). Subsequently, expression was seen in the telencephalonand diencephalon/mesencephalon boundary (stage 30; FIG. 10K, W). In thespinal cord, expression was restricted to the dorsal most regionincluding the roof plate (FIG. 10J, L, V, X). These expression patternsin the central nervous system are essentially the same as Zic3 exceptfor a slight difference in the expression level along the anterior toposterior axis (Nakata et. al., supra).

However, the expression patterns of the three Zic genes varied insomites and eye vesicles. Both Zic1 and Zic2 were expressed in thesomites. The level of Zic1 expression was higher than that of Zic2, incomparison to the expression in neural tubes (FIG. 10G, S), and Zic2expression extended more ventrally than Zic1. (FIG. 10L, X). Incontrast, Zic3 expression in the somites was negligible (Nakata et. al.,supra). As to the expressions in eye vesicles, Zic2 was expressedwhereas Zic1 and Zic3 were not (FIG. 10H, T, Nakata et. al., supra).Zic2 expression in the eyes was restricted to the Ciliary marginal zoneof neural retina, with no expression in the lens (FIG. 10J, V).

These expression patterns, when compared to that of Zic3, show that eachof the three Xenopus Zic genes is involved in several developmentalprocesses including those of the nervous system and somites.

D. Zic1 and Zic2 Induce Neural and Neural Crest Tissues

The effects of the Zic1 or Zic2 overexpression on the embryos wasexamined. First, MT-Zic1 (Zic1 tagged with myc epitopes ) or MT-Zic2mRNA were injected into both blastomeres of 2-cell stage embryos (FIG.11A-F). Ectopic pigment cells appeared in these embryos (FIG. 11C-F).The appearance of ectopic pigment cells was also found in the MT-Zic3 orZic3 mRNA injected embryos (FIG. 11B, Nakata et. al., supra). However,the pigment cells induced by MT-Zic1 overexpression were apparently lessdense and less frequent than those induced by MT-Zic3 or MT-Zic2overexpression (FIG. 11) [Ectopic pigment cells were found in 0/10 ofMT-Zic1 (100 pg), 16/16 of MT-Zic1 (500 pg), 16/20 of MT-Zic2 (100 pg),19/19 of MT-Zic2 (500 pg) and 13/13 of MT-Zic3 (100 pg) injectedembryos]. Each of the Zic proteins was expressed at equivalent levels ineach Zic mRNA injected embryo, suggesting that each Zic protein may havedifferent pigment cell-inducing activities (FIG. 11I).

Next, MT-Zic1 (250 pg) or MT-Zic2 (125 pg) mRNA were injected into ablastomere of a 2-cell stage embryo and sectioned at stages 35-36.Thickening of the neural tubes and the ectopic presumptive mesenchymaltissue with ectopic pigment cells in the injected side of these embryoswas observed (FIG. 11G, H). The ectopic pigment cells were also found inanimal cap explants overexpressing Mt-Zic1 (8/12), or Mt-Zic2 (11/11)(FIG. 12). To clarify whether these pigment cells were melanocytesderived from neural crest, the animal cap explants derived from theembryos obtained by the mating between albino female and wild type maleswere used. In this case, the shapes of the pigmented cells appearing inthe explants could clearly be observed. As expected, the pigment cellshad elaborate processes which were typically found in the melanocytes(FIG. 12). In addition, a neural crest marker twist (Xtwi, Hopwood et.al., “A Xenopus mRNA related to Drosophila twist is expressed inresponse to induction in the mesoderm and the neural crest,” Cell 59,893-903 [1989]) was expressed in the mesenchymal tissue beneath theectopic pigment cells in embryos in which Zic1 or Zic2 wereoverexpressed as observed with Zic3 overexpression (Nakata et. al.,supra). These findings suggest that pigment cells expressed in Zic1 orZic2 injected embryos were derived from neural crest.

To examine whether Zic1 and Zic2 overexpression results in alterationsin cell fate, the expression of a neural marker (NCAM) (Kintner andMelton, “Expression of Xenopus NCAM RNA in ectoderm is an early responseto neural induction,” Development 99: 311-325 [1987]), a neural crestmarker (Xslu) (Mayor et. al., “Induction of the prospective neural crestof Xenopus,” Development 121: 767-777 [1995]), and an epidermal antigen(EpA) (Jones and Woodland, “Development of the ectoderm in Xenopus:tissue specification and the role of cell association and division,”Cell 44: 345-355 [1986]) were examined at an early neurula stage (stage14) (FIG. 13). The NCAM-expressing neural plate region increased in theZic1 or Zic2 mRNA injected side [8/8 of MT-Zic1 (250 pg), 10/14 ofMT-Zic2 (125 pg) injected embryos] (FIG. 13A, D). Xslu expression wasalso increased in the Zic1 or Zic2 mRNA injected side [6/9 of MT-Zic1(250 pg), 18/18 of MT-Zic2 (125 pg) injected embryos] (FIG. 13B, E). Incontrast, the expression of EpA was significantly reduced on the Zic1 orZic2 mRNA injected side [18/22 of MT-Zic1 (250 pg), 20/24 of MT-Zic2(125 pg) injected embryos] (FIG. 13C, F). These observations suggestthat misexpressed Zic1 or Zic2 altered epidermal cell fate to neural andneural crest cell fate.

Next, the expression of several marker genes in the animal cap explantsfrom Zic1 or Zic2 overexpressing blastula were examined (stage 9) (FIG.14). Zic1 and Zic2 overexpression induced NCAM, a neuronaldifferentiation marker (N-rubulin; Chitnis et. al., “Primaryneurogenesis in Xenopus embryos regulated by a homologue of thedrosophila neurogenic gene Delta,” Nature 375, 761-766 [1995]) and Xtwiexpression in the explants, as expected based on the above results. Inaddition, a mid-hindbrain junction marker (En2; Hemmati-Brivanlou et.al., “Cephalic expression and molecular characterization of XenopusEn-2,” Development 3: 715-724 [1991]), but not a spinal cord marker[HoxB9 (which is the same as Xlhbox6); Wright et. al., “The XenopusXlHbox6 homeo protein, a marker of posterior neural induction, isexpressed in proliferating neurons,” Development 109: 225-234 [1990])was induced by Zic1 or Zic2 overexpression. These findings indicate thatneural tissue generated by the Zic1 or Zic2 overexpression hascharacteristics of anterior neural tissue similar to those observed withZic3 overexpression. These inductions appeared to occur without mesoderminduction since no mesodermal marker (M. actin; muscle actin) wasinduced in this case.

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: M (molar); mM (millimolar); μM (micromolar); mol(moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); pmol(picomoles); g (grams); mg (milligrams); μg (micrograms); ng(nanograms); l or L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); and° C. (degrees Centigrade).

Example 1 Cloning of the Zic3 Gene

(1) Preparation of Poly(A+) RNA from Xenopus laevis

Neurula Eggs of Xenopus laevis (Hamamatsu Seibutsu Kyozai, ShizuokaPref.) were incubated artificially according to method of Newport et al.(Newport et al., “A major developmental transition in early Xenopusembryos: I. characterization and timing of cellular changes at themidblastula stage,” Cell 30:675-686 [1982]) to obtain embryos. Theembryo was dipped in 2% cysteine-HCl (pH 7.8) to remove the jelly coatand then cultured in 0.1× Steinberg's solution (60 mM NaCl, 0.67 mM KCl,0.34, mM Ca(NO₃)₂, 0.83 mM MgSO₄, 10 mM Hepes, pH 7.4), followed byrecovery of the neurula.

From the recovered neurula, total RNA was extracted according to AGPCmethod. Subsequently, poly(A+) RNA was separated and purified therefromusing Oligotex dT30 (Roche).

(2) Preparation of a cDNA Library

cDNA was synthesized using the poly(A+) RNA from (1) above and TIMESAVER cDNA Synthesis Kit (Pharmacia). Briefly, single-stranded cDNAfragments were synthesized using the poly(A+) RNA as a template,oligo(dT)₁₂₋₁₈ primers and a cloned mouse reverse transcriptase. Then,double-stranded cDNA fragments were synthesized using E. coli RNase Hand E. coli DNA polymerase.

The resultant double-stranded cDNA fragments were blunt-ended usingKlenow fragment (Nippon Gene). Thereafter, an adaptor having an EcoRIrestriction site at one end and a blunt end at the other end was ligatedto the cDNA fragments using T4 DNA ligase. After phosphorylation of theEcoRI restriction site with T4 polynucleotide kinase, the cDNA fragmentswere introduced into the EcoRI site in the multicloning site of A ZAP II(a phage cloning vector) using a commercial kit (ZAP II VECTOR KIT;Stratagene). Thus, the packaging of cDNA was performed. Subsequently,the resultant phage vector was transformed into E. coli XL-1 blue, ahost, to thereby prepare a cDNA library.

(3) Preparation of Primers Specific to the Amino Acid Sequence of theZinc Finger

Motif of the Zic Gene Family Primers were synthesized based on wellconserved amino acid sequences in the mouse zinc finger domain. Briefly,5′ primer, 5′-GAGAACCTCAAGATCCACAA-3′ (SEQ ID NO: 5) was synthesizedbased on Glu Asn Leu Lys Ile His Lys (SEQ ID NO: 3). The 3′ primer,5′-TT(C/T)CCATG(A/G)ACCTTCATGTG-3′ (SEQ ID NO: 6) was synthesized basedon His Met Lys Val His Glu Glu (SEQ ID NO: 4).

The synthetic oligonucleotides were chemically synthesized with anautomated synthesizer (Applied Biosystem).

(4) Preparation of a cDNA Probe for Clone Isolation by PCR

PCR was performed using the cDNA from (2) above as a template and the 5′and 3′ primers from (3) above. The composition of the PCR reactionsolution was as follows.

-   -   First strand cDNA solution 1 μl    -   Sterilized water 70 μl    -   10×PCR buffer 10 μl    -   25 mM MgCl₂ 6 μl    -   2 mM dNTP mix 10 μl    -   100 μM 5′ primer (sense) 1 μl    -   100 μM 3′ primer (antisense) 1 μl    -   5 U/μl Taq polymerase 1 μl

After the above reaction solution was thoroughly mixed, 50 μl of mineraloil was layered over the solution. PCR was performed in a DNA thermalcycler for 30 cycles, one cycle consisting of reaction at 94° C. for 1min, at 55° C. for 1 min and at 74° C. for 2 min. As a result, afragment of 208 bp was obtained. This fragment was labelled withα-³²P-dCTP using a random primer labeling kit (Takara) to obtain a cDNAprobe for clone isolation.

(5) Isolation of a Clone

The cDNA library obtained in (2) above was plated on 12 NYZ plates(Falcon) so that ca. 150,000 plaques would be formed per plate. Uponthis plate, a nylon filter Colony/Plaque Screen (Dupont NEN) was placedand fixed with 0.5 N NaOH aqueous solution. Next, hybridization wasperformed in a hybridization buffer (50% formamide, 1 M NaCl, 10%dextran sulfate, 1% sodium dodecyl sulfate, 100 μg/ml denatured salmonsperm DNA) containing the labelled probe from (4) above at 42° C. for 18hours.

One clone was obtained from this screening. XL1-Blue was co-infectedwith the resultant clone and a helper phage R408 (Stratagene) to therebycut out the cDNA insert from AZAP II to pBluescriptSK(−), which was thentransformed into XL1-Blue. As a result, one clone having an insert ofca. 2.4 kb was obtained.

(6) Determination of the cDNA Nucleotide Sequence

The nucleotide sequence of the clone obtained in (5) above was analyzedusing ABI PRISMum Dye Cycle Ready Reaction Kit (Perkin-Elmer) and afluorescent automated DNA sequencer (Applied Biosystems). As a result,the nucleotide sequence for Zic3 gene with a length of 2364 bases wasobtained (SEQ ID NO: 1). This cDNA had a deduced amino acid sequenceconsisting of 441 amino acids (SEQ ID NO: 2).

A homology search was performed against GenBank/EMBL nucleic aciddatabases using FESTA homology search program (Pearson et al., Proc.Natl. Acad. Sci. USA 85: 2444-2448 [1988]). As a result, the nucleotidesequence of the gene of the invention exhibited 76% homology to that ofmouse Zic3 gene (Aruga, J. et al., J. Biol. Chem. 271: 1043-1047[1996]). With respect to the amino acid sequence, the gene of theinvention exhibited 66% and 35% homology to other Zic (Aruga, J. et al.,J. Biol. Chem. 271: 1043-1047 [1996]) and opa (Benedyk et al., GenesDev. 8: 105-117 [1994]), respectively. Accordingly, the gene of theinvention was designated Xenopus Zic3 (also called the “Zic3 gene” or“Zic3”).

Example 2 Functions of the Zic3 Gene

(1) Analysis of the Expression Pattern of Zic3 in Xenopus Embryos

In order to elucidate the expression pattern of the Zic3 gene of theinvention in Xenopus embryos, whole mount in situ hybridizations wereperformed (Daniel, H. S. et al., J. Biochem. Biophys. Methods 31:185-188 [1996]).

A digoxigenin (DIG)-labelled RNA probe for hybridization was synthesizedaccording to the method of Harland (Harland, R. M., Methods in CellBiology 36: 685-694 [1991]). Briefly, RNA was synthesized from 2.5 μg ofthe cDNA clone containing the Zic3 gene obtained in Example 1 using RNApolymerase, followed by DIG labelling. After the resultant DIG-labelledRNA probe was treated with DNase, sodium acetate solution was addedthereto to give a final concentration of 0.5 M and then the probe wasethanol-precipitated with 3 volumes of ethanol. The resultant mixturewas micro-centrifuged for 5 min at 12,000 rpm to precipitate the probe,which was re-suspended in 20 μl of 80% formamide and stored at −20° C.

Also, Xenopus embryos cultured up to individual stages in the samemanner as described in section (1) in Example 1 were fixed with aformalin fixative and stored in methanol at −20° C. For the purpose ofin situ hybridization, the embryos were distributed into 5 ml screwvials. The vial was shaken on Nutator (Becton-Dickinson) at roomtemperature.

The embryo was dipped in 50% methanol/50% 0.1 M triethanolamine (TEA, pH7.5) for 5 min and then washed with TEA for 5 min. The embryo wasincubated for 16 min in a mixture of TFA and acetic anhydride mixed at aratio of 1 ml:5 μl. Subsequently, the embryo was dipped in a mixedsolution of TEA and 3.7% formaldehyde for fixation. Prehybridization wasperformed by adding thereto a prehybridization solution (50% formamide,5×SSPE, 5% SDS, 1 mg/ml Torula RNA) and allowing it to stand at 60° C.for 1 hr and 10 min. Then, the prehybridization solution was replacedwith a sufficient amount of a hybridization solution (obtained by addingthe DIG-labelled RNA probe to the prehybridization solution at 1 μg/ml).An overnight incubation was performed at 60° C. for hybridization.

After the hybridization, the embryo was washed twice with 2×SSCcontaining 50% formamide at 60° C. for 30 min each time. Subsequently,the embryo was rinsed with maleate buffer (MAB; 100 mM maleic acid, 150mM NaCl, pH 7.5) for 5 min and then blocked with MAB containing 2% BMB(Boehringer-Mannheim Blocking Reagent) for 1 hr. The embryo was furtherincubated in MAB containing 2% BMB and 20% thermally-treated sheep serumfor 1 hr. Next, anti-dioxigenin antibody-conjugated alkaline phosphatase(Boehringer-Mannheim) was added thereto at 0.5 μl/ml, and incubation wascontinued for another 4 hr at room temperature. The resultant embryo waswashed with MAB for 1 hr at least 5 times, and then dipped in alkalinephosphatase buffer (100 mM Tris, 50 mM MgCl₂, 100 mM NaCl, pH 9.5) for10 min at room temperature for equilibration.

Per ml of the above buffer, 4.5 μl of nitroblue tetrazolium solution(NTB; 75 mg/ml of 70% dimethylformamide) and 3.5 μl of5-bromo-4-chloro-3-indolylphosphoric acid (BCIP; 50 mg/ml ofdimethyformamide) were added. The resultant mixture was incubated toinduce coloring reaction. The coloring was terminated by adding TE (10mM Tris-HCl, 1 mM EDTA, pH 8.0). Re-fixing and mounting were carried outaccording to the method of Harland (Harland, R. M., Methods in CellBiology 36: 685-694 [1991]).

As a result, Zic3 expression was detected at early gastrula in thedorsal lip and in the prospective neural plate [FIG. 1A (stage 10.25)and B (stage 10.5)]. In FIG. 1, an arrow indicates the gastrula andarrowheads indicate the prospective neural plate.

As gastrulation proceeded, expression of the Zic3 gene of the presentinvention decreased in the dorsal lip and increased in the prospectiveneural plate [FIGS. 1B and C (stage 10.5)]. FIG. 1C shows a crosssection of the embryo shown in FIG. 1B.

In late gastrula, Zic3 expression diminished gradually in the centralregion (FIG. 1D, stage 12). At the neural plate stage (FIG. 1E, stage14), Zic3 was expressed strongly in the prospective regions ofmesencephalon and anterior rhombencephalon. Thereafter, Zic3 expressionbecame stronger in the anteriorneural holds, whereas that in the trunkneural folds remained weak (FIG. 1F, stage 16).

At early tailbud stage (FIGS. 1G and H, stage 20), Zic3 expressionbecame gradually restricted to the dorsal region of the forebrain(telencephalon and diencephalon), the midbrain and the hindbrain, andits expression was weak in the dorsal region of the trunk.

After mid-tailbud stage, Zic3 expression disappeared in thediencephalon, but additional expression could be definitely confirmed inthe lateral mesoderm of the tailbud region (FIG. 1I, stage 30). Thecross section through the head at stage 30 showed that Zic3 expressionwas restricted to the dorsal part of the neural tube (FIG. 1J). In FIG.1J, “nt” represents the neural tube; “e” represents eyes; “fg”represents fore-gut. From these results, it was found that Zic3 isexpressed in those regions which are closely related to earlyneurogenesis.

(2) RT-PCR Analysis of the Temporal Expression Profiles of Zic3 andVarious Neural Marker Genes

Because Zic3 is expressed in the prospective neural plate region duringgastrulation, the temporal expression profile of Zic3 was compared withthose of other neural marker genes.

The marker genes used in this experiment were genes coding for NCAM,N-tubulin and transcription factors XASH-3, XATH-3, X1POU2 and NeuroD[Lee, J. E. et al., Science 268: 836-844 (1995); Ferreiro, B. et al.,Development 120: 3649-3655 (1994); Turner et al., “Expression ofachaete-scute homolog 3 in Xenopus embryos converts ectodermal cells toa neural fate,” Genes Dev. 8:1434-1447 (1994); Takebayashi, K. et al.,EMBO J. 16:384-395 (1997); Witta, S. E. et al., Development 121: 721-730(1995); Chitnis, A. et al., Nature 375: 761-766 (1995); Kintner, C. R.et al., Development 99: 321325 (1987); Zimmerman, K. et al., Development119: 221-231(1993); Oschwald, R. et al., Int. J. Dev. Biol. 35:399-405(1991)].

These genes can be obtained by synthesizing primers from the nucleotidesequence of the relevant gene described in the above references, usinggenomic DNA from Xenopus or the like as a template and utilizing PCRtechniques known in the art to amplify the relevant gene.

Comparison of temporal expression profiles was performed by RT-PCR.Briefly total RNA was extracted separately from embryos at individualstages of egg, 8-cell, morula, blastula, gastrula, neurula and tailbud,and subjected to RT-PCR. As an indicator for RNA recovery ratio, HistoneH4 was used (Turner et al., Nucleic Acids Res. 10:3769-3780 [1980]). Asa positive control, sibling control embryos were used. As a control tocheck the absence of genomic DNA, PCR was performed without reversetranscription.

Specifically, each embryo of the above-indicated stage was suspended in100 μl of a denaturing solution (4 M guanidine thiocyanate, 25 mM sodiumcitrate, 0.1 M 2-mercaptoethanol, 0.5% N-lauroyl sodium sarcosine) in a1.5 ml microtube and shaken vigorously. To this suspension, 10 μl of 2 Msodium acetate (pH 4.0) was added and mixed thoroughly. Subsequently,100 μl of water-saturated phenol was added thereto and mixed. Next, 30μl of CIA (chloroform: isoamyl alcohol=49:1 by volume) and 1 μl ofEtachinmate (Nippon Gene) were added to the mixture and shakenvigorously. The resultant mixture was left stationary on ice for 15 min.After centrifugation at 4° C. at 15,000 rpm for 20 min, the resultantupper layer was recovered into a fresh tube. Next, 250 μl of ethanol wasadded to the tube, which was centrifuged at 4° C. at 15,000 rpm for 10min to thereby pellet the RNA. The supernatant was discarded, and thetube was air-dried. Next, 88 μl of sterilized water, 10 μl of 10×DNasebuffer (BRL), 1 μl of RNasin (Promega), and 1 μl of DNaseI (Takara) wereadded to the tube and reacted at 37° C. for 1 hr to thereby degrade theDNAs mixed therein. Subsequently, 100 μl of ethanol was added to thetube, which was centrifuged at 40° C. at 15,000 rpm for 10 min tothereby pellet the RNA.

The supernatant was discarded. After the tube was air dried, 100 μl ofsolution K (0.01 M Tris, 0.005 M EDTA, 0.5% SDS, pH 7.8) and 1 μl of 20mg/ml proteinase K solution were added to the tube and reacted at 37° C.for 1 hr to thereby degrade the proteins mixed therein. Next, 100 μl ofphenol/CIA were added to the tube and mixed. The resultant mixture wascentrifuged at 4° C. at 15,000 rpm for 10 min. Thereafter, 100 μl of CIAwas added further and mixed. The resultant mixture was centrifuged at 4°C. at 15,000 rpm for 10 min. The resultant upper layer was recoveredinto a fresh tube, to which 250 μl of ethanol was added. Next, the tubewas centrifuged at 4° C. at 15,000 rpm for 10 min to thereby pellet theRNA. The supernatant was discarded. The tube was air-dried and thenallowed standing at room temperature.

The resultant RNA sample was dissolved in 10 μl of DPEC-treated water(obtained by adding 0.2 ml of diethylpyrocarbonate to 100 ml ofdistilled water, shaking the mixture vigorously and autoclaving it), and3 μl of this solution was placed into a microtube. To the microtube, 1μl of 100 pmol/μl random hexamer (Takara) and 7 μl of sterilized waterwere added and mixed thoroughly. The resultant mixture was incubated at72° C. for 2 min and at 37° C. for 5 min. Subsequently, 4 μl of 5×RTbuffer, 0.1 M DTT, 2 μl of 5 mM dNTP mix, and 0.5 μl of mouse leukemiavirus (MMLV)-derived reverse transcriptase (BRL) were added thereto andmixed thoroughly. Next, a reverse transcription reaction was performedat 37° C. for 1 hr. Subsequently, the reaction solution was maintainedat 98° C. for 10 min to terminate the reaction. Thus, a solution of thefirst strand cDNA was obtained and stored at −20° C. until use for thePCR synthesis of the second strand.

PCR was performed using the first strand cDNA solution obtained above asa template. The composition of the PCR reaction solution was as follows.

First strand cDNA solution  1 μl Sterilized water 70 μl 10x PCR buffer10 μl 25 mM MgCl₂  6 μl 2 mM dNTP mix 10 μl 100 μM primer (sense)  1 μl100 μM primer (antisense)  1 μl 5 U/μl Taq polymerase  1 μl

After the above reaction solution was mixed thoroughly, 50 μl of mineraloil was layered over the solution. PCR was performed 25-36 cycles, onecycle consisting of thermal denaturation at 94° C. for 0.5 min,annealing at 55° C. for 0.5 min and extension at 72° C. for 1 min. Aftercompletion of the reaction, 4 μl of the reaction solution was subjectedto agarose gel electrophoresis to examine the amplified product. Thus,expression of each gene was investigated.

Zic3 (sense) 5′-TTCTCAGGATCTGAACACAT-3′ (SEQ ID NO:7) (antisense)5′-CCCTATAAGACAAGGAATAC-3′ (SEQ ID NO:8) XASH-3 (sense)5′-GGACTCTCGCCTTGTGGC-3′ (SEQ ID NO:9) (antisense)5′-GATATGTTCTTGTAATAGTCAGT-3′ (SEQ ID NO:10) XATH-3 (sense)5′-TGGACCTCAGGCCATGTTC-3′ (SEQ ID NO:11) (antisense)5′-GATGCTGAGTGGAGGTGTTA-3′ (SEQ ID NO:12) X1POU 2 (sense)5′-ACCCAACGACCACGTGGACCTG-3′ (SEQ ID NO:13) (antisense)5′-AGCTCATTGCAGGAGGTGTCTG-3′ (SEQ ID NO:14) NeuroD (sense)5′-GTGAAATCCCAATAGACACC-3′ (SEQ ID NO:15) (antisense)5′-TTCCCCATATCTAAAGGCAG-3′ (SEQ ID NO:16) NCAM (sense)5′-CACAGTTCCACCAAATGC-3′ (SEQ ID NO:17) (antisense)5′-GGAATCAAGCGGTACAGA-3′ (SEQ ID NO:18) IV-tubulin (sense)5′-ACACGGCATTGATCCTACAG-3′ (SEQ ID NO:19) (antisense)5′-AGCTCCTTCGGTGTAATGAC-3′ (SEQ ID NO:20) Histone H4 (sense)5′-CGGGATAACATTCAGGGTATCACT-3′ (SEQ ID NO:21) (antisense)5′-ATCCATGGCGGTAACTGTCTTCCT-3′ (SEQ ID NO:22)

The results are shown in FIG. 2. As is clear from FIG. 2, Zic3expression was detected in early gastrula (stage 10). With respect tothe expression of other neural marker genes, XATH-3 and NCAM weredetected at mid-gastrulastage; and NeuroD and N-tubulin were detected atlate gastrula stage. Although XASH-3 and X1POU 2 were detected also atearly gastrula stage, their expression was extremely weak compared toZic3 expression.

Zic3 was first detected in the prospective neural plate regionimmediately after neural induction (FIG. 1A). Therefore, the onset ofexpression of Zic3 and a neural inducer gene chordin which is known tobe expressed at an early stage of neurogenesis (Sasai, Y. et al., Cell79:779-790 [1994]) were precisely compared. This comparison was madewith the following techniques.

Xenopus eggs were artificially fertilized with sperms in a cultureplate, and the embryonic development of all the fertilized eggs wasallowed to proceed in a synchronized manner. After the artificialfertilization, the eggs were cultured at 23° C. for 6 to 10.5 hr. Next,embryos were collected and immediately frozen. Zic3 RNA was extractedfrom these samples in the same manner as described in Section (1),Example 1, and subjected to RT-PCR. For chordin, an RT-PCR was performedusing the following primers.

(SEQ ID NO:23) Chordin (sense) 5′-AACTGCCAGGACTGGATGGT-3′ (SEQ ID NO:24)(antisense) 5′-GGCAGGATTTAGAGTTGCTTC-3′

As a result, it was found that the onset of Zic3 expression is 7.5 hrafter the fertilization, whereas that of chordin is 7 hr after thefertilization (FIG. 3). The numbers indicated above the lanes representhours of cultivation after the artificial fertilization. Since the onsetof Zic3 expression is only 30 min later than that of chordin, it wasfound that, like chordin, Zic3 also induces the initial step of neuralinduction.

(3) Zic3 Expression-Inducing Mechanism

The ectoderm (animal cap) of Xenopus laevis oocyte can be neuralized byprolonged culture in dispersal (Grunz, H. et al., Cell Differ.Dev.28:211-218 [1989]; Godsave, S. F. et al., Dev. Biol. 134:486-490[1989]). This occurs because ectoderm cells are relieved fromneuralization repressors as a result of dispersion of the cells.

Next, whether Zic3 is induced in animal cap explants was examined.Briefly, an animal cap explant was dipped in a buffer without Ca²⁺ andMg²⁺ ions and pipetted lightly to thereby obtain dispersed cells. Thecells were cultured under such condition in a medium without Ca²⁺ andMg²⁺ ions at 23° C. for 4 hr. Next, the cells were allowed to form acell mass again, and the mass was cultured up to a time point equivalentto the neurula stage.

Subsequently, the expression of Zic3 and other genes (epidermal keratin,NCAM, Xtwi, Xslu and Histone H4) was tested by RT-PCR. Briefly, RNA wasextracted from non-dispersed animal cap explants and dispersed animalcap explants. As primers for individual genes, the above-mentionedprimers and those described below were used. A series of RT-PCRs wereperformed under the same conditions as in Section (1) in this Example(animal cap assay).

Epidermal keratin (sense) 5′-CACCAGAACACAGAGTAC-3′ (SEQ ID NO:25)(antisense) 5′-CAACCTTCCCATCAACCA-3′ (SEQ ID NO:26) Xtwi (sense)5′-AGTCCGATCTCAGTGAAGGGCA-3′ (SEQ ID NO:27) (antisense) 5′-TGTGTGTGGCCTGAGCTGTAG-3′ (SEQ ID NO:28) Xslu (sense)5′-GCCCTATTTCCTTGTTGC-3′ (SEQ ID NO:29) (antisense)5′-AACCCTTCTTGGTTGCAC-3′ (SEQ ID NO:30)

!

The results are shown in FIG. 4A. In each lane, “Intact” representsanimal cap explants (non-dispersed cells); “Dispersed” represents animalcaps cultured in dispersal; “Embryo” represents embryos; and “RT_”represents the results without reverse transcriptase. Zic3 expressionwas not detected in intact animal cap explants, though the expression ofepidermal keratin (an epidermal marker gene) was detected on the otherhands, expression of Zic3 and a neural marker gene NCAM was detected indispersed cells, but expression of epidermal keratin was not detected.

The neuralization that occurs in animal cap-derived dispersed cells isconsidered to be due to the attenuation of BMP4-mediated signals whichinduce ectodermal cells into epidermal cells (Wilson, P. A. et al.,Nature 376:331-333 [1995]). Therefore, the following experiment wasperformed on the assumption that Zic3 expression can actually be inducedin vivo by blocking the BMP4-mediated signals, although an understandingof the mechanism is not necessary to practice the present invention andthe present invention is not limited to any particular mechanism.

Briefly, a dominant negative form of BMP receptor (dnBMPR) mRNA (Suzuki,A. et al., Devlop. Growth. Differ. 37:581588 [1995]) was injected intoembryos to over-express dnBMPR therein. Next, Zic3 expression in earlygastrula stage embryos was examined by in situ hybridization in the samemanner as described above.

Specifically, Zic3 mRNA and dnBMPR mRNA were synthesized by in vitrotranscription. Zic3 mRNA (100 pg) was injected into a two-cell stageembryo with a glass microneedle. As a control, LacZ mRNA-injected earlygastrula was used. dnBMPR mRNA (500 pg) was injected into the ventralregion of a two-cell stage embryo, which was developed in 1× Steinberg'ssolution containing 5% Ficoll up to the early gastrula of stage 10.25.The resultant embryo was subjected to whole mount in situ hybridizationwhich was performed in the same manner as described above to therebyexamine Zic3 expression.

As a result, in both the dnBMPR mRNA-injected embryo and the dnBMPR mRNAnon-injected embryo, Zic3 expression was observed at naturally expectedsites (arrowheads, FIG. 4B). In the dnBMPR mRNA-injected embryo,expression of ectopic Zic3 was induced in the ventromarginal zone of thegastrula (arrows, FIG. 4B). This shows that injection of dnBMPR mRNAcauses expression of excessive dnBMPR in cells, which in turn inhibitsthe BMP4 signals and induces the neuroectoderm (Zimmerman, L. B. et al.,Cell 86:599-606 (1996)]. Thus, it was found that Zic3 expression can beinduced in vivo by blocking the BMP4 signals.

(4) Zic3 Overexpression Test in Early Embryos

The expression pattern of Zic3 in Xenopus and its activity to regulateneural induction suggest that Zic3 plays some role in early steps ofneurogenesis. Thus, the function of Zic3 was examined by overexpressionexperiment in embryos. First, Zic3 mRNA was injected into one blastomereof two cell stage embryos so that Zic3 would be overexpressed in theleft or right hemilateral body alone.

Briefly, Zic3 mRNA, LacZ mRNA and dnBMPR mRNA were synthesized by invitro transcription in the same manner as described above. Zic3 mRNA wasinjected into one blastomere of two-cell stage embryos or twoblastomeres of eight-cell stage embryos independently or in combinationwith LacZ mRNA. The dnBMPR mRNA (500 pg) was injected into the ventralregion of two-cell stage embryos. For the animal cap assay, Zic3 mRNAwas injected into the animal hemisphere of the two blastomeres oftwo-cell stage embryos. As a control, embryos injected with LacZ mRNAalone or H₂O alone or non-injected embryos were also tested. Injectedembryos were cultured in 1× Steinberg's solution containing 5% Ficolluntil mid-blastula stage. Next, they were transferred into 0.1×Steinberg's solution and subjected to animal pole assay at stage 9 or insitu hybridization at various stages in the same manner as describedabove.

The results are shown in FIG. 5. FIG. 5, panel A shows Zic3 mRNAuninjected control side of the embryo (stage 27); panel B shows Zic3mRNA injected side (stage 27). Panel C shows a dorsal view of theanterior region of Zic3 mRNA injected embryo (stage 36). Panels D-F aremicroscopic photographs of transverse sections of the embryo shown inpanel C. Panels G-J show overexpression of Zic3 when 100 pg of Zic3 mRNAor control LacZ mRNA was injected into two blastomeres of eight-cellstage embryos. In panel G (stage 36), mRNA was injected into twodorsoanimal blastomeres; in panels H-J (H, I: stage 25, J: stage 20),mRNA was injected into two ventroanimal blastomeres. In panels G and H,upper figures show lateral views of control LacZ mRNA injected embryos;and lower figures show lateral views of Zic3 mRNA injected embryos.Panel I shows higher magnification of the clusters in panel H. Panel Jshows Xtwi expression in the embryos injected with LacZ mRNA or Zic3mRNA into ventroanimal two blastomeres at eight-cell stage.

In almost all cases, the head side of the Zic3 mRNA injected embryos wasenlarged and exhibiting poorly formed eyes (FIG. 5B, C), whereas Zic3mRNA uninjected embryos exhibited normally formed eyes (FIG. 5A, C).

The sections through the head region of the Zic3 mRNA injected embryosshowed that neural walls were considerably thickened in the injectedside (FIG. 5D-F). In addition to this change in neural walls,presumptive mesenchymal tissue, which may derive from the neural crestin the cephalic region, showed a remarkable hyperplasia. In most cases,however, neural retinas were considerably distorted and lesshyperplastic (FIG. 5D). Additionally, retinal pigment cells diminishedand, in particular, lenses were not induced at all (FIG. 5C, D).Further, eye abnormalities were observed in the embryos injected withZic3 mRNA into dorsal blastomeres (FIG. 5G, lower). Remarkable clustersof ectopic pigment cells appeared in the embryos injected with Zic3 mRNAinto ventral blastomeres (FIG. 5H, lower). In contrast, no suchabnormalities were observed in Zic3 mRNA uninjected embryos.

On the other hand, Xtwi expression was observed in the head neural crestof the control embryo (FIG. 5J, upper, arrowhead). In contrast,expression of ectopic Xtwi was induced near the ectopic clusters ofpigment cells in the ventral side of Zic3 mRNA injected embryos (FIG.5J, lower, white arrowheads). The expansion of the Xtwi expressingcephalic neural crest (FIG. 5J, lower, black arrowheads) was observed ineight-cell stage embryos, and ectopic clusters of pigment cells wereobserved in the cephalic region (FIG. 5B).

Subsequently, Zic3 mRNA was injected into two dorsoanimal orventroanimal blastomeres of eight-cell stage embryos to express Zic3restrictedly at the dorsal or ventral side (FIG. 5G-J). This experimentwas performed in the same manner as described above using 80 embryos.

When Zic3 mRNA was injected into dorsoanimal blastomeres, heads of theembryos were enlarged, and the eyes showed abnormalities in theneuroepithelium of retina, diminishing of retinal pigment cells and lossof lens (these changes are the same as observed in the embryos injectedat two-cell stage) (58/80 embryos tested) (FIG. 5G). In the anteriorregion, neural tube closure was delayed. Pigment cells were found in thedorsal head (58/80 embryos tested).

In contrast, when Zic3 mRNA was injected into ventroanimal blastomeres,clusters of ectopic pigment cells appeared in the ventral epidermis(90/111 embryos tested). The clusters of the ventral pigment cells werearrayed remarkably on the ridge of the hyperplastic tissue whichtransverses the ventral side. These pigment cells were considered to bemelanocytes which are derived from the neural crest. Therefore, in situhybridization of Zic3 mRNA injected embryos using a neural crest markerXtwi as a probe was performed. The in situ hybridization techniques usedwere the same as described in (1) above.

As a result, ectopic Xtwi expression was observed near the ectopicallyappearing clusters of pigment cells in addition to the expansion of theXtwi-expressing region in the cephalic neural crest.

(5) The Role of Zic3 in Early Embryogeny

In order to examine how the overexpression of Zic3 alters cell fate inearly stage embryos, the inventors tested the expression of NCAM, Xtwi,Xslu and EpA (an epidermal antigen gene) (Jones, E. A. & Woodland, H.R.: Cell 44:345-355 [1986]) in early neurulas.

Briefly, a total of 100 pg of Zic3 mRNA was injected into a blastomereof two-cell stage embryos. The expression patterns of the above geneswere examined at stage 14 by in situ hybridization using NCAM and Xsluprobes and by immunohistochemical staining using EpA monoclonal antibody(obtained from Mr. E. Jones of Univ. of Warwick, UK).

The results are shown in FIG. 6. In FIG. 6A-D, any of the panels shows adorsal view of a stage 14 embryo. Panel A, panel B and panel C show theresults when NCAM, Xtwi and Xslu were used as a probe, respectively.Panel D shows the results when EpA monoclonal antibody was used.

NCAM expression increased markedly in the anterior neural plate regionof the Zic3 mRNA injected side (31/45 embryos tested) (FIG. 6A). Xtwi(43/45 embryos tested) and Xslu (12/12 embryos tested) expression inneural crest cells was also increased by the injection (FIGS. 6B and C).However, in the Zic3 mRNA injected site of the epidermis, EpA stainingwas decreased (FIG. 6D).

If it is assumed that epidermal fate changes into neural and neuralcrest fate as a result of the injection of Zic3 mRNA, the epidermisshould be reduced at the site of Zic3 mRNA injection. To test thispossibility, the expression of EpA in Zic3 mRNA injected embryos wasdetermined as described below. Briefly, embryos were dipped in Dent'sfixative (20% dimethyl sulfoxide, 80% methanol) and shaken gentlyseveral times. Next, they were left at −20° C. overnight for fixation.After removal of the fixative, a bleach (10% H₂O₂, 47% methanol, 20%DMSO) was added to the embryos, which were then left standing for 1 to 2days for bleaching. After removal of the bleach, 100% methanol was addedto the embryos, which were then stored at −20° C. Thereafter, theembryos were washed with TBS (50 mM Tris-HCl (pH 7.5), 150 mM NaCl,0.05% Tween 20) for 20 min twice. On the other hand, EpA monoclonalantibody was diluted to 1/100 to 1/1000 with TBS containing 20% normalgoat serum. Using the resultant dilution, the embryos were primarilystained at 4° C. overnight. Subsequently, the embryos were washed withTBS for 1 hr 5 times. Next, using ca. 200-fold dilution of peroxidaseconjugated secondary antibody in TBS containing 20% normal goat serum,the embryos were secondarily stained at 4° C. overnight. After 1 hrstaining with TBS 5 times, the embryos were secondarily stained with acoloring solution (TBS containing 0.5 mg/ml diaminobenzidine and 0.02%H₂O₂). After a color of an appropriate density was formed, the embryoswere washed with 100% methanol for 10 min twice. Subsequently, theembryos were dipped in BABB solution (benzyl alcohol:benzylbenzoate=1:2)to make them transparent, followed by storing in 100% methanol.

As a result, expression of EpA was significantly reduced in the Zic3injected site (FIG. 6D). This fact indicates that Zic3 alters epidermalcell fate into neural and neural crest cell fate.

(6) Function Expression Test for Zic3

The above studies suggest that Zic3 plays important roles in earlyneural and neural crest development. In order to examine how Zic3 actsin these processes, the expression of several marker genes were testedby RT-PCR in Zic3 mRNA injected animal cap explants.

One hundred pg of Zic3 or LacZ mRNA (control) was injected into two-cellstage embryos. The animal cap of each embryo was explanted at stage 9and cultured. When the sibling embryos reached stage 20, the expressionof neural marker genes (NCAM, Neurogenin, NeuroD, XASH-3, XATH3, X1POU2) and neural crestmarkers (Xtwi, Xslu) was examined by RT-PCR. Also,the expression of an early mesodermal marker Xbra (Xenopus brachyury)and a dorsal mesodermal marker M. actin (muscle actin) was examined byRT-PCR when the sibling embryos reached stage 10.5 and stage 20,respectively, in the same manner as describe above. For Neurogenin, Xbraand M. actin, an RT-PCR was performed using the following primers.

(SEQ ID NO:31) Neurogenin (sense) 5′-CAAGAGCGGAGAAACTGTGT-3′ (SEQ IDNO:32) (antisense) 5′-GAAGGAGCAACAAGAGGAAG-3′ (SEQ ID NO:33) Xbra(sense) 5′-GTCCGTACACTCACAGAAAC-3′ (SEQ ID NO:34) (antisense)5′-GAGGTGTAGAGCCAAGTAAG-3′ (SEQ ID NO:35) M. actin (sense)5′-GCTGACAGAATGCAGAAG-3′ (SEQ ID NO:36) (antisense)5′-TTGCTTGGAGGAGTGTGT-3′

The results are shown in FIG. 7. Zic3 induced all of the neural andneural crest marker genes tested. Although uninjected (Uninj.) or LacZinjected (LacZ) caps did not express any of these markers, animal capsinjected with Zic3 mRNA (Zic3) expressed all of the neural and neuralcrest marker genes tested (FIG. 7A). However, Zic3 did not inducemesodermal markers (FIG. 7B).

These results demonstrate that Zic3 is able to direct the induction ofneural tissues except mesoderm and that Zic3 is able to change directlythe epidermal fate of cells to neural and neural crest fate. Further,the expression of a molecular marker En-2 expressed in anterior neuralplate (Hemmati Brivanlou, A. et al., Development 111:715-724 [1991]) anda posterior marker X1Hbox6 (Wright, C. V. E. et al., Development109:225-234 [1990]) was tested by RT-PCR in the same manner as describedabove. For En-2 and X1Hbox6, an RT-PCR was performed using the followingprimers.

(SEQ ID NO:37) En-2 (sense) 5′-CACAAGGGGTTAAAGGCAAG-3′ (SEQ ID NO:38)(antisense) 5′-CCCAGTGTCTCTCTCAGTAT-3′ (SEQ ID NO:39) XlHbox6 (sense)5′-TACTTACGGGCTTGGCTGGA-3′ (SEQ ID NO:40) (antisense)5′-AGCGTGTAACCAGTTGGCTG-3′

As a result, though the anterior neural marker En-2 was induced, theposterior marker X1Hbox6 was not induced (FIG. 7C). This result isconsistent with the previous finding that the neural tissue generated bythe blockage of BMP4 signals is anterior neuroectoderm(Hemmati-Brivanlou, A. et al., Cell 77:283-295 [1994]; Sasai, Y. et al.,Nature 376:333-336 [1995]; Lamb, T. M. et al., Science 262:713-718[1993]). Therefore, it has become clear that Zic3 has an activity toinduce anterior neuroectoderm.

Zic3 overexpression induced the neural marker NCAM and the neural crestmarkers Xtwi and Xslu in explants (FIG. 7A). This presents a contrast tothe result that Zic3 was expressed, but Xtwi and Xslu were not, indispersed animal cap cells (FIG. 4).

From the results described above, it has been shown that Zic3 inducesthe so-called proneural genes and that Zic3 acts upstream of theseproneural genes. Therefore, Zic3 has neurogenesis-inducing activity, andin particular, early neurogenesis-inducing activity and thus, can becalled a master gene for neural induction.

Example 3 Isolation of Xenopus Zic1 and Zic2 cDNA Clones

Xenopus neurula (stage 17) cDNA was subjected to 30 cycles of PCR (at94° C., 1 min.; 55° C., 1 min and 74° C., 1 min, respectively) (Nakataet al., 1997). The 5′ primer was a 5′-GAGAACCTCAAGATCCACAA-3′ (SEQ IDNO: 5), derived from ENLKIHK (SEQ ID NO: 3); sequence based on zincfinger domain of the mouse Zic family genes), and the 3′ primer was5′-TT(C/T)CCATG(A/G)ACCTTCATGTG-3′ (SEQ ID NO: 6), which was the reversetranslation of HMKVHEE (SEQ ID NO: 4). A 208 bp PCR product wassequenced. The fragment was used to screen a lambda ZAP cDNA libraryprepared from Xenopus neurula embryos (Pharmacia Biotech; TIME SAVERcDNA Synthesis Kit, Strategene; Lambda ZAP^(R) II Vector Kit) under lowstringency conditions. Two cDNA clones were isolated (Zic1, 1.8 kb:Zic2, 2.9 kb) and these were auto-sequenced by ABI PRISM Dye PrimerCycle Sequencing Ready Reaction Kit (Perkin-Elmer).

Example 4 Plasmid Construction

The Zic1 open frame reading (275-1825, pZic1) (end-filled) fragment wascloned into the StuI site of the pCS2+vector (Turner and Weintraub,1994) (pCS2+Zic1). The full-length Zic2 coding region was cloned intothe EcoRI-XbaI site of the pCS2+vector by PCR amplification of pZic2(452-1957) (pCS2+Zic2).

The Zic3 open reading frame (EcoRI [-10: that lies in the vectorimmediately 5′ to the end of the cDNA]-StuI [1460]; pZic3) fragment wascloned into the EcoRI and StuI sites of the pCS2 vector (pCS2+Zic3). Thefull-length coding region of each Zic gene family was also clonedin-frame into the EcoRI-XbaI site of the pCS2+MT (myc-tag) vector(Turner and Weintraub, 1994) by PCR amplification from the original cDNAphagemid (pCS2+MT-Zic1, pCS2+mT-Zic2, and pCS2+MT-Zic3).

Example 5 Embryo Manipulations

Xenopus laevis were purchased from Hamamatsu Seibutsu Kyozai, (Shizuoka,Japan). Embryos were obtained by artificial fertilization (Newport andKirschner, 1982). The jelly coats were removed by immersing the embryosin 2% cystein-HCl (pH 7.8). Embryos were cultured in 0.1×Steinberg'ssolution and staged according to Nieuwkoop and Faber (Nieuwkoop, P. D.,Faber, J., “Normal Table of Xenopus laevis (Daudin) North-Holland,Amsterdam [1967]).

Microinjection was carried out as previously described (Moon andChristian, “Microinjection and expression of synthetic mRNAs in Xenopusembryos,” Technique 1: 76-89 [1989]). mRNA for injection was synthesizedby in vitro transcription. Xenopus Zic1, Zic2 or Zic3 mRNA was injectedwith or without LacZ mRNA (a gift from Dr. A. Muto) into one or twoblastomeres of 2-cell stage embryos or two blastomeres of 8-cell stageembryos. For animal cap assay, mRNA was injected into the animal side oftwo blastomeres of 2-cell stage embryos. Injection of LacZ mRNA alone orno injection was done as a control in these experiments. Injectedembryos were cultured in 5% Ficoll in 1× Steinberg's solution. Theembryos were replaced in 0.1× Steinberg's solution at midblastula stageand were subjected to animal cap assay at stage 9 or whole-mount in situhybridization at various stages.

For preparation of animal cap explants, 6 animal caps were dissectedfrom Xenopus Zic1, Zic2 or Zic3 mRNA injected or uninjected embryos in1×MMR at stage 9 and cultured in 0.5×MMR.

Example 6 RNA Isolation and RT-PCR Assay

Preparation of total RNA and RT-PCR assay were carried out as previouslydescribed (Nakata et al., 1997). Histone H4 or EF-1a was used to monitorRNA recovery. Sibling control embryos served as positive controls. PCRwas performed with RNA that had not been reversed-transcribed to checkfor DNA contamination. Some primer sequences were obtained from TheXenopus Molecular Marker Resource (See e.g. websitevize222.zo.utexas.edu). In addition, the following primers were used:

Zic1: 5′-ATGAAGGTCCACGAAGCATC-3′ (SEQ ID NO:45)5′-CGTGCTGTGATTGGACGTGT-3′ (SEQ ID NO:46) Zic2:5′-ACGGCAGCGTTATCTCCTAG-3′ (SEQ ID NO:47) 5′-TATACACCGAGGGAGGCATC-3′(SEQ ID NO:48)

Example 7 Histology and Whole-mount in situ Hybridization

Whole-mount in situ hybridization was performed essentially as describedpreviously (Chitnis et al., 1995; Shain and Zuber, “Sodium dodecylsulfate (SDS)-based whole-mount in situ hybridization of Xenopus laevisembryos,” J. Biochem. Biophys. Methods 31, 185-188 [1996]) usingdigoxigenin-labeled antisense probes for Zic1, Zic2, NCAM (Kinter andMelton, 1987), and Xslu (Mayor et al., 1995). Some stained embryos werethen embedded in paraplast and sectioned at 5 μm using a microtome.Whole-mount immunohistochemistry was performed essentially as describedpreviously using EpA monoclonal antibody (Jones and Woodland, 1986).

Example 8 Western Blotting

Pools of 20 embryos were homogenized in lysis buffer containing 10 mMTris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.1 mM PMSF and 0.1 mMleupeptin. Proteins were separated by 7.5% SDS-polyacrylamide gelelectrophoresis and immunoblotted with the anti-c-myc antibody (9E10,Santa Cruz Biotechnology) diluted 1:1000 (0.1 mg/ml).

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular and cellular biology, biochemistry, or related fields areintended to be within the scope of the following claims.

1. An isolated nucleotide sequence encoding a protein having the aminoacid sequence set forth in SEQ ID NO: 44, wherein the protein is aneurogenesis inducing protein.
 2. The isolated nucleotide sequence ofclaim 1, wherein the nucleotide sequence comprises SEQ ID NO:
 43. 3. Arecombinant vector comprising the nucleotide sequence of claim
 1. 4. Anisolated cell comprising the recombinant vector of claim
 3. 5. A methodfor producing a neurogenesis-inducing protein, comprising the steps of:a) providing: i) the recombinant vector of claim 3, and ii) a host cell;b) introducing said recombinant vector into said host cell to produce atransformed cell which contains said recombinant vector and expressessaid neurogenesis-inducing protein; and c) culturing said transformedcell to produce said neurogenesis-inducing protein.
 6. The method ofclaim 5, further comprising the step of: d) isolating said neurogenesisinducing protein.
 7. The recombinant vector of claim 3, wherein saidvector comprises in operable linkage a promoter and a vector backbone,wherein said vector backbone is a viral nucleotide sequence selectedfrom the group consisting of adenovirus, adeno-associated virus, herpesvirus, vaccinia virus and retrovirus.
 8. A composition comprising therecombinant vector of claim 3 and liposomes.